CN111032090A - Patch implant composition for cell implantation - Google Patents

Abstract

The present invention provides compositions and methods for transplanting cells into a solid organ by an implantation strategy. These methods and compositions are useful for repairing diseased organs or for modeling disease states in experimental hosts. The method involves attaching a patch implant, a "bandage-like" covering, to the surface of a tissue or organ, the "bandage-like" covering containing epithelial cells as well as mesenchymal cells during supportive early lineage. The cells are incorporated into a gel-forming biomaterial that is prepared under serum-free, defined conditions that collectively support the sternness of the donor cells. The implant is covered with a biodegradable, biocompatible, bioresorbable substrate for immobilizing the implant at a target site. The cells in the implant migrate to and throughout the tissue such that they are evenly dispersed within the recipient (host) tissue within two weeks. The mechanism of donor cell implantation and integration into the organ or tissue involves multiple membrane-associated and secreted forms of MMPs.

Description

Patch implant composition for cell implantation

Cross Reference to Related Applications

This patent application claims priority from us patent application No. 62/518,380 filed on 12.6.2017 and us patent application No. 62/664,694 filed on 30.4.2018 in accordance with clause 119 (e) of the american codex 35, the contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates generally to the field of cell transplantation or tissue implantation. And more particularly to implantation from, and in particular to implantation into, a solid organ or tissue. The present invention relates to compositions and methods for providing strategies for rapid transplantation, implantation and integration of cells into solid organs and tissues to treat diseases or conditions of the solid organs or tissues, or to establish model systems for diseases. A representative example of this potential is cell therapy for the treatment of liver or pancreatic diseases.

Background

There has long been a need for implantation strategies for cells from solid organs, other than for hematopoietic cell transplantation or for mesenchymal stem/progenitor cell transplantation. Turner, R. et al, transplantation90,807-810 (2010); gattinini, L. et al Nature Medicine 23,18-27 (2017); trousnon A. et al, CellStem Cell 17,11-22 (2015); sun B.K. et al Science 346,941-945 (2014); lainas, P. et al JHEPATAL 49, 354-. Transplantation of hematopoietic and mesenchymal cells is typically performed by cellular delivery through the vascular groove and, due to microenvironment signaling (a process called "homing"), depends on activation of adhesion molecules in the transplanted cells when in the relevant target site. The method for skin (using a similar method for an ocular target) employs an implantation method in which cells are applied directly to the target site. Sun B.K. et al Science 346,941-945 (2014). Many methods of implantation for the skin are available for cells from solid internal organs, but require extensive modification to adapt the cells to the microenvironment of these internal organs. The implant must counter the mechanical forces exerted by the interaction of the tissue and organ with each other; examples include the effect of the lungs during respiration, or the compression of the diaphragm by the liver, or the transient effect of mechanical forces exerted by the intestinal tract on adjacent tissues during food processing. Implants, especially those for internal organs, are challenging to design due to issues with respect to size, shape, and complexity of the organ structure in addition to significant dynamic mechanical forces.

For decades, cell therapy for cells from solid organs other than skin has been attempted using transplantation via the vascular route or by direct injection into tissues. When delivered by any of these strategies, most transplanted cells die or are transported to ectopic sites where they can survive for months and form tissue at inappropriate sites, potentially causing clinical adverse reactions. Turner, R. et al transfer 90,807-810 (2010); lanzoni, G, et al, Stem Cells 31,2047-2060 (2013). Implantation of the liver can be improved by coating the cells with hyaluronic acid and delivering them to the liver via the blood vessels; the improvement in implantation efficiency is due to the natural clearance process of hyaluronic acid by the liver. Nevi et al Stem Cell Research & Therapy 8,68, 2017. However, these improvements are still not as effective as the improvements of the implantation strategy and, importantly, still allow delivery of cells to ectopic sites.

There remains a need for improved methods of implantation of cells into solid organs. The present disclosure satisfies this need and provides related advantages.

Disclosure of Invention

For a long time, there is a continuing need for implantation strategies for cells from solid organs (Turner, r. et al, Transplantation90,807-810 (2010)), other than for hematopoietic cell, mesenchymal stem cell Transplantation or for skin. Transplantation of hematopoietic and mesenchymal cells is typically via the vascular sulcus and depends on activation of adhesion molecules in the relevant target site due to microenvironment signaling, a process called "homing". The method for skin uses an implantation method in which cells are applied directly to a target site.

Transplantation of cells from solid organs other than the skin has long been used for vascular delivery. This is illogical, as the adhesion molecules on these cells are always active and lead to rapid (a few seconds) cell aggregation that can produce life-threatening emboli. Even with successful embolization control to minimize health risks, cell engraftment efficiency is low (only-20%) for adult cells and even lower (< 5%) for stem/progenitor cells. Most transplanted cells die or are transported to ectopic sites where they can survive for months and form tissue at inappropriate sites, potentially causing clinical adverse reactions. A small fraction of the cells implanted at the target site slowly integrate, taking weeks to months to become a significant part of the tissue. Implantation in the liver would be improved if the cells were coated with hyaluronic acid and delivered via the blood vessels due to hyaluronic acid clearance of the tissue (e.g. of the liver). (Nevi et al, StemShell Research & Therapy 8,68, 2017).

Applicants propose a distinct approach, one that was found to be even more successful than coating cells with hyaluronic acid: implants are placed directly on the surface of a target site and when they are under conditions that enhance the transplanted biological material, the unique phenotypic traits of the transplanted biological material and certain cells are used. This is similar in some respects to the strategy for cell therapy of the skin, but requires substantial modification to the internal organs due to mechanical action, wear or compression of organs in close proximity to each other, and due to the unique fluid microenvironment around a particular organ and the size, structure and complexity of the organs.

Described herein are novel patch implant compositions and methods for the transplantation of cells to tissues and solid organs. In some embodiments, the methods and implants are applicable to internal organs, and their design characteristics depend on the level of maturation of the cells, particularly whether the cells are stem cells or mature cells. In some embodiments, disclosed herein are donor cells (optionally autologous or allogeneic cells) for patch implants, optionally incorporated into the implant biomaterial as a cell mixture or organoid form, aggregates of epithelial stem cells, and their natural lineage-appropriate mesenchymal cell partner, e.g., mesenchymal stem/progenitor cells, such as early lineage mesenchymal cells (ELSMCs). In some embodiments, the donor cell is a somatic cell that is incorporated into the implant material as a cell suspension of somatic epithelium and is paired with mesenchymal stem/progenitor cells (optionally ELSMCs) in a ratio designed to optimize the expression of membrane-associated and/or secreted Matrix Metalloproteinases (MMPs) of the stem/progenitor cells. In some embodiments, other important variables are the implantation of the biomaterial and the substrate material, which are required to be neutral in their effect on the differentiation of the donor cells.

Aspects of the present disclosure relate to a patch implant for retaining and maintaining a single cell population or a mixed population of cell populations, the patch implant comprising (a) a single cell type or a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), or comprising MMPs from another source (e.g., purified or recombinant MMPs), the cell population or mixed population being supported in a culture medium present in a hydrogel matrix having sufficient viscoelastic properties to allow migration of the mixed population (optionally, within or away from the hydrogel and/or within or away from the patch implant); (b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in the direction of the substrate; and optionally (c) a hydrogel overlaid on a serosal (i.e., exterior) surface of the substrate, the serosal surface being opposite the surface contacting the mixed population and, in embodiments in which the patch implant is tethered to the target site, opposite the side contacting the target site (e.g., organ or tissue). In some embodiments, the layer prevents or inhibits adhesion by, or with, other tissues or organs. In some embodiments, the patch implant is configured to maintain and maintain the mixed population while inhibiting differentiation or further maturation of the at least one early lineage stage cell type to a late lineage stage that is no longer capable of expressing membrane-associated and/or secreted MMPs. The patch implant may be a single layer plus a substrate or multiple layers.

In some embodiments, the substrate is porous or non-porous. In some embodiments, the substrate comprises a porous mesh, scaffold, or membrane. In some embodiments, the substrate comprises a filament; a synthetic fabric; or natural materials (such as amnion, placenta or omentum); or a combination thereof. In some embodiments, the substrate comprises a porous mesh infused with a hydrogel. In further embodiments, such infusion prevents migration of cells away from the target organ or tissue. In some embodiments, the substrate comprises a solid material.

In some embodiments, one or more of the hydrogels comprises hyaluronic acid.

In some embodiments, the culture medium comprises a Kubota's medium or another medium that supports stem cells and is capable of maintaining a dry quality.

In some embodiments, the mixed population comprises mesenchymal cells and epithelial cells. In some embodiments, the epithelial cells can be ectodermal, endodermal, or mesodermal cells. In some embodiments, the mesenchymal cells comprise Early Lineage Stage Mesenchymal Cells (ELSMCs). In some embodiments, the ELSMC includes one or more of a hemangioblast, a precursor of endothelium, a precursor of stellate cells, and a Mesenchymal Stem Cell (MSC). In some embodiments, the epithelial cells comprise epithelial stem cells. In some embodiments, the epithelial cells comprise biliary stem cells (BTSCs). In some embodiments, the epithelial cells comprise committed and/or mature epithelial cells. In some embodiments, the committed and/or mature epithelial cells comprise mature parenchymal cells. In some embodiments, the mature parenchymal cells include one or more of hepatocytes, cholangiocytes, and islet cells. In some embodiments, the mesenchymal cells and epithelial cells both comprise stem cells.

In a certain embodiment, the mixed population comprises autologous cells and/or allogeneic cells.

In some embodiments, one or more cell types are genetically modified.

Additional aspects relate to methods of using the disclosed patch implant compositions. Accordingly, provided herein is a method of implanting cells into a target tissue, the method comprising, consisting of, or consisting essentially of contacting the target tissue with the patch implant disclosed above.

In some embodiments of the method, the target tissue is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, gastrointestinal tract, lung, prostate, breast, brain, bladder, spinal cord, skin and subcutaneous dermal tissue, uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and skeletal tissue. In some embodiments of the method, the target tissue is liver tissue. In some embodiments of the method, the target tissue is pancreatic tissue. In some embodiments of the method, the target tissue is biliary tissue. In some embodiments of the method, the target tissue is a gastrointestinal tract tissue. In some embodiments, the tissue is diseased, damaged, or has a disorder. In some embodiments of the method, the target tissue is kidney tissue.

In some embodiments of the method, the target tissue is an organ. In some embodiments of the method, the organ is an organ of the musculoskeletal system, digestive system, respiratory system, urinary system, female reproductive system, male reproductive system, endocrine system, circulatory system, lymphatic system, nervous system, or integumentary system. In some embodiments of the method, the organ is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, stomach, intestine, lung, prostate, breast, brain, bladder, spinal cord, skin and subcutaneous dermal tissue, uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and bone. In some embodiments, the organ is diseased, damaged, or has a disorder.

In some embodiments of the method, the liver disease or disorder is liver fibrosis, cirrhosis, hemochromatosis, liver cancer, biliary atresia, nonalcoholic fatty liver disease, hepatitis, viral hepatitis, autoimmune hepatitis, fascioliasis, alcoholic liver disease, α 1-antitrypsin deficiency, glycogen storage disease type II, transthyretin-associated hereditary amyloidosis, Gilbert's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, bade-Chiari syndrome, liver trauma, or Wilson's disease.

In other aspects, provided herein are methods of treating a subject having a pancreatic disease or disorder, comprising, consisting of, or consisting essentially of contacting a pancreas of the subject with the patch implant disclosed above. In some embodiments of the method, the pancreatic disease or disorder is diabetes, pancreatic exocrine insufficiency, pancreatitis, pancreatic cancer, Oddi (Oddi) sphincter dysfunction, cystic fibrosis, pancreatic division, cricoid pancreas, pancreatic trauma, or pancreatic ductal hemorrhage.

In other aspects, provided herein are methods of treating a subject having a disease or disorder of the gastrointestinal tract, comprising, consisting of, or consisting essentially of contacting one or more intestines of the subject with a patch implant disclosed above. In some embodiments, the gastrointestinal disease or disorder is gastroenteritis, gastrointestinal cancer, ileitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, peptic ulcer disease, celiac disease, fibrosis, vascular dysplasia, hirschsprong's disease, pseudomembranous colitis, or gastrointestinal trauma.

In some aspects, provided herein are methods of treating a subject having a kidney disease or disorder, the method comprising, consisting of, or consisting essentially of contacting one or more kidneys of the subject with a patch implant disclosed above. In some embodiments of the method, the kidney disease or disorder is nephritis, nephropathy, nephritic syndrome, nephrotic syndrome, chronic nephropathy, acute kidney injury, kidney trauma, cystic nephropathy, polycystic nephropathy, glomerulonephritis, IgA nephropathy, lupus nephritis, kidney cancer, Alport (Alport) syndrome, amyloidosis, Goodpasture's syndrome, or Wegener's granulomatosis.

Drawings

Fig. 1A-1D provide information about porcine donor cells for patch implants. Fig. 1A is a schematic illustration of the process and estimated time required to prepare the organoid, assemble the patch implant and perform the surgery. In fig. 1B, donor cells for stem cell patch implants were isolated from cell suspensions from biliary tissue of transgenic pigs; the cells were made organoid in serum-free bottom tower medium and on low-adherence culture dishes. Biliary stem cells (BTSCs) and their early lineage mesenchymal cell (ELSMC) partners, angioblasts, and organoids of endothelial and stellate cell precursors. They are shown in phase contrast micrographs in comparison to cells displaying transgenic, Green Fluorescent Protein (GFP) expression. ). All cells of the aggregate were green in color, as the transgene was present in both epithelial and mesenchymal cells. The transgene is coupled to the histone (H-2B) locus. Paraffin embedding, sectioning and histology of stem cell organoids stained with hematoxylin/eosin. (d) Enlargement of organoids of BTSC and ELSMC. Figure 1C shows Immunohistochemistry (IHC) showing expression of stem cells, liver and pancreatic markers, indicating that these cells are precursors to both liver and pancreas. The IHC assay identifies the outer layer by a metaphase stem cell marker, such as EpCAM, and identifies the inner cells that express very primitive genes, such as pluripotency genes and endoderm transcription factors (e.g., SOX17, SOX9, PDX 1). Figure 1D is a representative qRT-PCR assay to assess expression of various genes in organoids, indicating that the cells are stem cells or early progenitor cells. Controls were mature hepatocytes from piglet liver.

Fig. 2A-2F provide information about the major components of the patch implant. Fig. 2A is a schematic illustration of a patch implant fixed to a pig liver, the right figure being the composition of the implant. Early lineage stage cells of both epithelial and mesenchymal cells are the source of Matrix Metalloproteinases (MMPs) that are key regulatory factors for the production of engraftment. The matrix component of the implant biomaterial on which the donor cells are placed is soft (-100 Pa) and is not (or minimally) sulfated, such as a hyaluronic acid hydrogel. The structure of the implant is made up of a layer of biomaterial and cells tethered to the target site. The media components are free of serum, growth factors and cytokines that affect donor cell differentiation and should be tailored for survival and expansion of cells at the early lineage stage (such as stem/progenitor cells). The substrate has sufficient tensile strength for surgical procedures, but is neutral in its effect on donor cell differentiation (cells in which type I collagen should be avoided). The substrate dip is coated with a thicker 10 × hydrogel (-700 Pa) to act as a barrier to direct migration of donor cells to the target tissue and minimize adhesion. After attachment to the target site, a 2 × HA hydrogel (sufficiently fluid to coat or coat on the serosal surface) is added and used to further minimize adhesion. Fig. 2B depicts an implant secured to the liver or pancreas of a host. Fig. 2C is a schematic view of an implant showing the layers that make up the implant composition. Figure 2D depicts the results of an assay that empirically evaluates the rheological or viscoelastic properties (shear and compressive mechanical forces) of a particular hydrogel layer. Figure 2E provides a formulation of the viscoelastic properties of a 3-layer hydrogel. Fig. 2F is a close-up view of a patch implant sutured to the surface of a pig liver.

Fig. 3A-3D depict Immunohistochemical (IHC) and histological results of liver patch implants. Fig. 3A shows the trichromatic dyeing results at one week of the patch implant. Trichrome recognises collagen (blue), cytoplasm (red) and nucleus (black) and is used to recognise the Glisson's capsule (usually adjacent the surface of the liver lobule) and adhesions (at the serosal surface of the implant). There is a high level of blue staining in the layer at the serosal surface, which means adhesion to the implant. Additionally, the implant separates from the host tissue at the interface between the substrate and the host; this is often the case because of the large number of MMPs generated at this interface. The remodeling zone provides evidence of loss of the classical lobular structure of the liver; they create regions: wherein donor cells migrate to the tissue and alter host tissue architecture in parallel. In the low magnification image (a), trichrome staining of the implant placed on the liver confirmed that extensive remodelling of the gleason bursa occurred and generally led to separation between the implant and the host liver. In the high magnification image (b), the remodelling zone is very broad and consists of a region (c) near the implant where the liver leaflet structure is completely missing and a region (d) within the remaining liver leaflets which is destroyed during the remodelling process. Fig. 3B shows the trichromatic staining results at three weeks of patch implant. The hyaluronic acid in the implant has been resorbed, leaving only the substrate (a). With resorption of HA, the glisson bursa reappears (b) and the liver lobules near the implant stabilize again to their typical histological pattern, such as lobules and acini of the liver. (b) The arrow in (b) indicates the re-appearance of collagen during the re-formation of the Gleason's capsule. Fig. 3C and 3D show hematoxylin/eosin staining results of sections from the implant one week after implantation (C) and two weeks after implantation (D). The upper graph is 40X. The parts in the figures (a, b, c) are enlarged views enlarged by 100 ×; the rectangular chart below each of these magnifications is magnified 200 x. The figure shows 3 sites of the implant: (a) a site within the substrate and associated implant biomaterial; (b) a site at the interface between the implant and the host tissue; and (c) sites within the liver lobules. Hematoxylin/eosin staining yields images that help understand the implantation and migration processes that combine the characteristics of inflammatory processes.

Figures 4A-4C show the fate of implantation, migration and rapid maturation to adults within a week. Fig. 4A is a low magnification view of a patch implant on the surface of a pig liver after one week. The dashed line represents the interface between the implant and the host liver. Donor GFP + cells were visualized by labeling with antibodies to GFP and a secondary antibody conjugated to a Red fluorescent probe Novo Red (the nucleus was pink; white arrows indicate regions with large numbers of donor GFP + stem cells). Staining of the nuclei blue with 4, 6-diamidino-2-phenylindole (DAPI) enables identification of host cells with blue nuclei only and donor cells with pink nuclei (merged images of DAPI and Novo Red). Host tissue (a) hyaluronic acid extending to the implant (HA, black background); tissue near the basement occasionally contains organoids (insets), but most donor cells are dispersed in single cells; a large number of dispersed donor GFP + stem cells were visible throughout the host tissue (b). There is no evidence of the presence of the Gleason's capsule in this region constituting the remodeled region. FIG. 4B shows that implantation and migration of donor cells is rapid; within one week, all donor cells were in the host liver; there were donor cells both near the implant site and on opposite sides of the liver lobe (estimated distance from the implant was at least 1.5 cm). An ongoing study is to analyze the more distant regions of piglet liver (i.e. other lobes of the liver) to more accurately define how far donor cells can migrate within a defined period of time. Donor cells (pink nuclei) near the host mature hepatocyte lobule (forest green autofluorescence from lipofuscin) on the side of the liver lobes remote from the implant site are shown. Figure 4C shows the fate of donor cell maturation to adults that occurs in parallel with reabsorbed HA. An enlargement of the region (a) containing donor GFP + cells (single cells with pink nuclei) near the host hepatocytes (forest green in color (autofluorescence of lipofuscin)), which are readily distinguishable from the hepatocytes of mature donor source (b) (light purple in color) (combined images of pink-GFP, blue-DAPI, and green-lipofuscin), that is, they are lineage restricted from donor GFP + stem cells. By other IHC assays (data not shown), the leuchun green cells in the plates of both host and donor hepatocytes were demonstrated to be endothelial and stellate cells.

Fig. 5A-5C compare the engraftment and maturation of cells in liver patch implants one and two weeks after transplantation. Fig. 5A is an examination of pig liver 1 week after patch implantation. Immunohistochemistry of pan-cytokeratin (pCK) and Sox9 was performed on consecutive 3- μm sections using collagen-stained azo dye sirius red dye; and Immunofluorescence (IF) staining. At the patch implant site, the implanted donor cells coalesce with the liver lobules. In the upper panel (original magnification ═ 5 ×), the patch implant is composed of mesenchymal and epithelial pCK⁺Cells (arrows). In the middle panel, a higher magnification (20 ×) is provided. Epithelial cells show a typical immunophenotype of biliary stem cells (BTSCs) expressing biliary cytokeratin (pCK) and the endodermal stem cell marker Sox 9. The BTSCs within the patch implant are arranged into cell strings that reassemble the bile canaliculus (arrows) and maintain direct continuity with the hepatocyte plates of the adjacent liver lobules (wedge symbols). Host hepatocytes in the lobules were pCK and Sox9 negative. In the lower panel (original magnification ═ 20 ×), immunofluorescence of GFP allowed the identification of single implanted cells and their progeny. Hepatocytes in the leaflets adjacent to the patch implant were GFP positive, indicating that these cells are donor cell-derived cells that have substantially merged with the host liver. At the interface between the patch implant and the liver leaflets, pCK⁺/GFP⁺GFP in tubules (i.e. biliary epithelial cells of donor origin) and leaflets⁺/pCK^-Cells (donor-derived hepatocytes) maintained direct continuity (wedge symbols), indicating that the engrafted cells matured into the lives of hepatocytesAnd (6) carrying out transportation. Fig. 5B is an examination of pig liver 2 weeks after patch implantation. IF staining revealed GFP⁺Cells are present within leaflets remote from the implantation site they are evenly dispersed so they are in a mixture of host cells (cells with blue nuclei from DAPI) and donor cells (blue and GFP-labeled red pooled pink/purple nuclei of DAPI) they co-express mature hepatocyte markers such as Hepatocyte Nuclear Factor (HNF)4 α (mixture of green and pink/purple nuclei) and albumin (green cytoplasm and pink/purple nuclei) individual or pooled channels are included nuclei are shown in blue (DAPI) original magnification: 40 x fig. 5C is an assessment of pig liver one week after patch implantation showing a region of broad remodeling at the interface between patch implant and host tissue the sections in the low magnification map and the enlarged leaflets of fig. 1 are hematoxylin/eosin (light staining), the sections in fig. 2 are stained with Vector-SG providing blue sections/grey, the sections in blue section stain fetal protamine protein, the background is hematoxylin/eosin, 5 is a section of hematoxylin the liver, and the sections in which are shown by Vector-SG, provide blue sections of stained for which the blue section/grey, the background is indicative of the presence of a change in the donor cell status of a, such as a sign of a change in the donor cells, and thus the status of the absence of the presence of a marker of a host cell in the mature liver.

Fig. 6A-6D provide information about a patch implant of stem cell organoids tethered to the pancreas fig. 6A is a low magnification (panoramic scan) map of GFP + donor cells implanted into the submucosa (region containing the bunner's glands) of the majority of the pancreas and duodenum fig. GFP (green), insulin (red), DAPI (blue) donor-derived GFP + cells appear near the site where the implant is located and show integration into the pancreatic parenchyma in the patch implant site fig. 6B shows that the donor cells mature into functional islets at high magnification fig. 6B shows that donor cells are from donor-derived β cells (yellow-from the combined color of GFP and insulin-stained red) are observed at high magnification in combination with host/insulin + (red) cells in the pancreatic parenchyma (yellow-from the combined color of GFP and insulin-stained red) β in combination with host/insulin-derived from the pancreatic parenchyma, and the high magnification map of the extracellular secretion of pancreatic cells from the pancreatic parenchyma, the high magnification of the starch-derived from the parenchyma of the kidney cells, the patch implant cells are shown in a continuous magnification map of the presence of GFP (blue) and the high magnification of the focus of the starch-derived from the parenchyma of the kidney cells in the pancreas, the patch implant, the patch, the high magnification map shows that the absence of the kidney cells in the kidney cells and the high magnification of the patch.

FIGS. 7A-7H provide a characterization of Matrix Metalloproteases (MMPs). MMPs consist of calcium-dependent, zinc-containing enzymes that solubilize a large gene family of extracellular matrix components. At least 24 isoforms are known in pigs, one subset of which are secreted factors (e.g., MMP1, MMP2, MMP7, MMP9) and another subset of which are membrane-associated factors (e.g., MMP14, MMP 15). MMP1 was identified by IHC, particularly in the remodeled region, but could not be identified by RNA-seq, as the annotated form of porcine MMP1 has not been available for RNA seq analysis. Fig. 7A, 7B, 7C, and 7D show that isoforms of secretory and membrane-associated species are expressed by both stem/progenitor cells and mature cells. Quantification of expression levels indicated that the membrane-associated forms were similar for both stem/progenitor and mature cells (note the comparison in fig. 7D). In contrast, the secreted form is expressed at very high levels in stem/progenitor cells and at low or negligible levels in mature cell types. The cell population of adult cells analyzed was isolated from a suspension of piglet liver and biliary tissue and consisted of CD45+ cells (hematopoietic cells), CD146+ cells (stellate cells), CD31+ cells (endothelium), EpCAM +/CD 45-cells (adult diploid hepatocytes and biliary epithelium). These EpCAM +/CD 45-cells are mature parenchymal cells present in the livers of piglets. BTSCs were isolated from biliary lines by the protocol given in the examples. Figure 7E shows representative MMP expression in the remodeled region with BTSC/ELSMC implant. In sections of the patch implant adjacent to the BTSC/ELSMC, staining was performed with a trichrome representing the remodeled area (bracket). This region appears as a linear band in red and blue, with cellular and matrix components undergoing lysis by the MMP "sea". The strip ends at the edge of the leaflet, which is largely still intact, but begins to "wear out" at the boundary due to the action of MMPs derived from invading cells. FIG. 7F shows a representative image of an IHC assay for MMP1(Novo-red +). Methyl green is background staining. The lobular/acinar structures of the liver are solubilized in the wavy coil of the cell and are marked by the strong expression of MMP1 (the secreted isoform of MMP). FIG. 7G shows sections stained for MMP2(Novo-red +). Hematoxylin is background staining. The lobular/acinar structure of the liver has disappeared and has been replaced by a mixture of cells with strong staining for MMP2 (russet). Fig. 7H shows the ongoing remodeling process within the liver lobules. Liver lobules have become bands of cells scattered among invading cells; MMP2+ expression (rust color) was very high and contributed to the loss of leaflet/acinar structure. With clearance of hyaluronic acid (2-3 weeks), leaflet structure reappears.

Fig. 8 is a schematic illustration of the implantation and integration phenomena in the liver and on the pancreas.

Fig. 9A-9E provide information about a patch implant of mature (adult) hepatocytes collocated with Mature Mesenchymal Cells (MMC), such as endothelial or stellate cells. These patch implants cannot be implanted. Implantation is achievable if hepatocytes are paired with early lineage mesenchymal cells (ELSMCs), here porcine Mesenchymal Stem Cells (MSCs). Implantation occurs if present with an ELSMC, but is limited to the area near implantation. Figure 9A shows trichrome staining of normal pig liver. The bars are 200 μm for the low magnification plot (a) and 50 μm for the high magnification plot (b). Note the boundary between collagen in the grignard capsule and the liver acinus. Fig. 9B shows trichrome staining of a normal adult hepatocyte patch implant paired with Mature Mesenchymal Cells (MMC), endothelium and stellate cells, which was not implantable. In the low magnification plot (a), note that the Gleason's capsule is intact and the cells are still on top of the capsule. (b) At high magnification, there is evidence that the cells have some degree of remodeling (plasticity phenomenon) in the lobules next to the implant (a patchy red color within the hepatocytes). This plasticity is thought to be due to membrane-associated MMPs known to be present on both stem and adult cells. Fig. 9C shows IHC assay for a patch implant of normal adult hepatocytes collocated with Mature Mesenchymal Cells (MMC). Sections were stained with antibodies to RBMY-1, hematoxylin as a counterstain. The Gleason's capsule is intact, as are the boundary regions between the leaflets. At high magnification (b), apparently no implantation occurred. (d) A negative control for non-specific staining (staining without primary antibody) is indicated. Fig. 9D shows the trichrome staining of a normal adult hepatocyte paired with ELSMC patch implant, here a porcine Mesenchymal Stem Cell (MSC) that functions as a cell source for MMPs. The implant separates at the interface between the implant and the host tissue. Brackets indicate remodeling regions. Note that the liver leaflets have lost the stroma that normally constitutes the boundary zone between them and wear occurs at the edges. In high magnification (a) donor cells are visible throughout the image (light red, while donor cells in the center of the leaflet are dark red); (b) in (b) is an enlarged view showing the area under the patch and in (c) where the gleason bursa is significantly thinner (compared to the area to the left of the box). There is significant extensive remodeling in cells adjacent to the implant (c). Fig. 9E shows a patch implant of hepatocytes collocated with ELSMCs (porcine MSCs) after one week. Sections (a) were stained with antibodies to RBMY-1 (brown) and methyl green as counterstain. The donor cells engraft (rust red areas) and mature into adult parenchymal cells in the acini near the implant. Section (b) shows a magnified view of the image near the rest of the thinned grignard capsule showing that the donor cells (dark brown nuclei) are evenly dispersed in the host cells (nuclei are methyl green in color). Section (c) is a negative control for (b). Sections (d) were stained with antibodies against GFP (coupled with Novus red, producing russet brown) with methyl green as counterstain. Most of the cells have been engrafted and formed a deep red band of donor (mature) hepatocytes within the host hepatic acinus. The Gleason's capsule remained but the thickness was reduced. No migration beyond the liver region near the implant was observed within the three week time frame of the experiment.

Fig. 10 is a schematic comparing the implantation of stem cells with adult cells.

FIG. 11 shows evidence that the implantation process involves migration of cells a considerable distance within the host tissue. Organoids one week after transplantation for BTSC/ELSMC implants are shown here. A schematic of the liver divided into 8 different regions is used to represent the regions that were used to assess the presence of donor cells. Sections were prepared from regions 1-8 and then stained to identify donor cells. The table summarizes the findings, which show the distance between the implant and each zone and the replication of the GFP + cells present. The image on the left of the table is a scan of a representative slice from each region. Dark brown staining was strongest in 6 near the implant and became lighter with increasing distance from the implant, zone 1 was lightest.

FIGS. 12A-12E provide evidence of donor cell migration throughout the host liver. GFP + cells were stained with Novo-red (russet brown); host cells were stained with methyl green. Fig. 12A is a low magnification view of the interface of the implant and the host liver. Separation of the implant from the host liver is usually visible (note that this is also true in fig. 3); and have been shown to be associated with abnormally high levels of secreted MMPs. Enlarged views of regions (a) and (b) are given below. Note the areas in the low magnification plot and in the magnified plot of (b) where the staining was speckled and these areas showed a hazy appearance and were confirmed due to the hyaluronic acid level in the tissue. Fig. 12B depicts the intermediate zone to which the cells migrated. The donor cells are in the entire tissue in both the bile duct and acinar parenchyma. FIG. 12C shows the distant zone to which the cells migrate. Note that only bile ducts were stained. Fig. 12D provides an enlarged view showing donor cells in the bile duct. FIGS. 12E and 12F provide essentially enlarged views showing donor cells with GFP markers in the nucleus.

Fig. 13 shows the ill-conditions obtained for a patch implant with certain substrates (see also table 1 and table 2). These include necrosis, adhesions, and finding sites where cholestasis occurs when the implant is placed too close to some tubes so that swelling causes the tubes to become blocked.

Fig. 14 shows a graph of two lineage stages for epithelial cells (fig. 14A) and mesenchymal cells (fig. 14B) and corresponding biomarker profiles.

FIG. 15 shows organoids of kidney implanted H2B-GFP + BTSC/ELSMC patches. Evaluation was performed 1 week after implantation. Panel a shows trichrome staining of implanted kidneys. The kidney was prepared in a transection fashion to expose the deeper layers with the "V" shaped implant. The lower half "V" stained bright blue is the implanted side on the kidney; the upper half "V" of the figure is a layer deeper than the implanted layer. Panel B shows H & E staining for the same section of implanted kidney. Panel C is a higher magnification view of a patch implanted kidney. The renal capsule under the implant relaxes in a manner similar to that in the liver (from dissolution by MMPs). Panel D shows IHC staining of GFP + cells (dark red) implanted in the kidney at the layer under the patch. Panel E shows the implantation of GFP + cells (dark red) in the deeper layers of the kidney. Necropsy reports indicated the absence of necrosis in the implanted kidney or elsewhere in the animal receiving the patch implant.

Description of the attached tables

Table 1 provides a summary of surgical or other methods for a patch implant.

Table 2 provides a comparison of substrates tested against exemplary patch implants.

Table 3 provides a summary of the antibodies used in the examples for IHC and IF.

Table 4 provides a summary of the primers used for the qRT-PCR assay.

Detailed Description

Embodiments in accordance with the present disclosure are described more fully hereinafter. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this patent application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Although not explicitly defined below, such terms should be interpreted according to their usual meaning.

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds (2012) Molecular Cloning, A Laboratory Manual, 4 th edition; ausubel et al eds (2012) Current Protocols in Molecular Biology series; methods in Enzymology series (Academic Press, Inc., N.Y.); MacPherson et al (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al (1995) PCR 2: A practical Aproach; harlow and Lane eds (2014) Antibodies, A Laboratory Manual, 2 nd edition; freshney (2011) Culture of Animal Cells A Manual of Basic Technique, 6 th edition; gait, eds (1984) Oligonucleotide Synthesis; U.S. Pat. nos. 4,683,195; hames and Higgins eds (1985) nucleic Hybridization; anderson (1999) Nucleic Acid Hybridization; hames and Higgins eds (1984) Transcription and transformation; immobilized Cells and Enzymes (IRL Press (1986)); perbal (1984) A Practical Guide to Molecular Cloning; miller and Calos eds (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring harbor laboratory); makrides eds (2003) Gene Transfer and Expression in Mammarian Cells; mayer and Walker eds (1987) biochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al (1996) Weir's Handbook of Experimental Immunology.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present disclosure also contemplates that, in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustration purposes, if the specification states that the complex comprises components A, B and C, it is specifically intended that either of A, B or C, or a combination thereof, may be omitted and discarded, either individually or in any combination.

All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and vary by increments (+) or (-) of 1.0 or 0.1, or by +/-15%, or 10%, or 5%, or 2%, as the case may be. It is to be understood that all numerical designations are preceded by the term "about," although this is not always explicitly stated. It is also to be understood that, although not always explicitly indicated, the reagents described herein are exemplary only and that equivalents of such reagents are known in the art.

Definition of

As used in the description of the invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, the term "about," when referring to a measurable value, such as an amount or concentration (e.g., the percentage of collagen in the total protein in a biomatrix scaffold), etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

When used to describe the selection of any component, range, dosage form, etc., the terms or "acceptable", "effective" or "sufficient" disclosed herein mean that the component, range, dosage form, etc., is suitable for the purposes disclosed.

Also as used herein, "and/or" means and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in an alternative manner ("or").

As used herein, the term "comprising" means that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of … …" (and grammatical variations) should be interpreted as encompassing the recited or steps, as well as "those material steps that do not materially affect one or more of the basic and novel features of the recited embodiments. See, Inre Herz,537F.2d 549,551-52,190U.S.P.Q.461,463(CCPA1976) (all highlighted above); see also MPEP § 2111.03. As used herein, the term "consisting essentially of … …" should not be construed as equivalent to "comprising". "consisting of … …" shall mean excluding more than trace amounts of other ingredient elements and the essential method steps for administering the compositions disclosed herein. Aspects defined by each of these transitional terms are within the scope of the present disclosure.

As used herein, the term "patch implant" refers to a composition of cells embedded or contained in a suitable biological material to allow for the transplantation of donor cells (allogeneic or autologous) to a host. In some embodiments, the term refers to a composition of cells embedded or contained in an appropriate biological material to allow for the transplantation of donor cells to a host. The biomaterial is a biomaterial that can be prepared under specified conditions (e.g., culture medium of purified free fatty acids complexed with purified albumin-plus-lipoprotein carrier molecules such as high density lipoproteins) including (optionally cured) in a soft gel (less than 200Pa, optionally about 100Pa) and covered with a substrate having sufficient tensile strength to allow surgical attachment or tethering to the host's tissues or organs, but with chemical properties that have minimal impact on donor cell differentiation, optionally supplemented with basal media and/or nutrient factors, vitamins, amino acids, carbohydrates, minerals, insulin, transferrin/Fe, and/or lipids. Supplements containing factors that may drive differentiation of cells, especially mesenchymal cells during Early Lineages (ELSMCs), should be avoided; these factors include serum, growth factors and cytokines that affect ELSMC, as well as mature matrix components (e.g., type I collagen).

As used herein, the term "substrate" refers to a material that is a substrate or barrier on the surface of the patch implant that is capable of tethering the implant to the target site and/or facilitating the migration of cells therein to the target site and/or preventing or inhibiting the migration of cells to the substrate. The substrate is or includes a "biodegradable, biocompatible material", "biocompatible, biodegradable material" or any variant thereof, referring to such materials: (i) is biocompatible with respect to the subject to whom the material is transplanted, (ii) exhibits mechanical elasticity to withstand compressive and shear forces (particularly one of the interior) occurring on organs and tissues, which in turn allows the material to function as surgical tissue, and (iii) has a neutral or minimal effect on the state of differentiation of cells contacting the material. In some embodiments, the substrate of the patch implant comprises such a material. In such embodiments, the mechanical elasticity of (ii) should be such that the substrate can tether the implant to the target site. In further such embodiments, the substrate directs migration of cells toward the target site-for example, by affecting differentiation of those cells that migrate in a direction away from the target site or by physically impeding the migration. In this regard, suitable materials include, but are not limited to, Seri-Silk (optionally profiled Seri-Silk or derivatives thereof), amniotic membrane (e.g., the fetal side-facing amniotic membrane) or extracts thereof, and/or patches or fabrics composed of PGA and/or PLLA. Non-limiting examples of suitable synthetic material patches include woven patches composed of 91% PGA-9% PLLA copolymer, woven patches composed of 91% PGA-9% PLLA copolymerWoven patch or a nonwoven patch consisting of 100% PGA. More generally, suitable substrates may include silk forms of silk moth (such as Seri)^RSurgical silk stents (Sofregen, New York, NY)), other derivatives of silk moth and synthetic fabrics (such as polyglycolic acid-poly-L-lactic acid copolymer (PGA/PLLA) forms).

In some embodiments, the substrate is also bioresorbable. As used herein, "bioresorbable" refers to a material that can be broken down by the body of the host or recipient of the implant and does not require mechanical removal. In some embodiments, the bioresorbable substrate is bioresorbable in the range of about 2 to about 10 weeks, about 2 to about 20 weeks, about 2 to about 52 weeks, about 4 to about 16 weeks, about 4 to about 12 weeks, or about 4 to about 8 weeks. In some embodiments, the bioresorbable substrate is coated with a coating for about 4 to about 8 weeks; is bioresorbable in the range of about 4 to about 12 weeks, about 4 to about 16 weeks, about 4 to about 20 weeks, and about 4 to about 52 weeks.

As used herein, the biomaterial of the implant and the biomaterial of the implant independent of the substrate include biomaterials that can form a hydrogel. The term "gel" refers to a solid, gelatinous material that can have properties ranging from soft and weak to hard and tough. A gel is defined as a crosslinked system that is substantially dilute and exhibits a non-flowing state when in a steady state. Gels are mostly liquids by weight, however, they behave like solids due to the three-dimensional cross-linked network within liquids. It is the cross-linking within the fluid that gives the gel structure (stiffness, consistency, mechanical or viscoelastic properties) and contributes to the adhesiveness of the gel. Thus, a gel is a dispersion of liquid molecules within a solid, where the solid is the continuous phase and the liquid is the discontinuous phase. "hydrogel," also referred to herein as a "hydrogel matrix," is a non-limiting example of a gel composed of a macromolecular polymer gel constructed from a network of polymer chains. Hydrogels are synthesized from hydrophilic monomers or hydrophilic dimers (e.g., in the case of hyaluronic acid) by chain growth or step growth and network formation. The network structure and void defects enhance the ability of the hydrogel to absorb large amounts of water via hydrogen bonding. Thus, hydrogels produce the characteristic strong yet elastic mechanical properties. When old bonds within a material break, they are able to undergo spontaneous formation of new bonds. The structure of the hydrogel and electrostatic attraction drive the formation of new bonds through non-covalent hydrogen bonds.

Biomaterials used in implants have mechanical properties, consistency, which can be more strictly defined as the viscoelastic properties of the biomaterial. See https:// en. wikipedia. org/wiki/Viscoelasticity. Implant Biomaterials to facilitate implantation must be very soft (e.g., about 100Pa), allowing the donor cells to remain in immature conditions (Lozoya et al, Biomaterials 2011; 32(30):7389 and 7402), so membrane-associated and/or secreted forms of MMP can be produced.

As used herein, the term "viscoelastic" refers to the property of a material that exhibits both viscous and elastic characteristics when undergoing deformation. Viscous materials are able to resist shear flow like honey and are linearly strained over time when stress is applied. Elastic materials are strained when stretched and quickly recover their original state once the stress is removed. Viscoelastic materials have elements of both properties and therefore exhibit time-dependent strain. Elasticity is generally a result of stretching of bonds along crystalline planes in ordered solids, while viscosity is a result of diffusion of atoms or molecules within the amorphous material. While there are many instruments that test the mechanical and viscoelastic responses of materials, Broadband Viscoelastic Spectrometers (BVS) and Resonant Ultrasonic Spectrometers (RUS) are more commonly used to test viscoelastic behavior because they can be used above and below ambient temperatures and are more specific for testing viscoelastic properties. Both instruments employ damping mechanisms at various frequencies and time ranges, without requiring time-temperature superposition. The use of BVS and RUS to study the mechanical properties of a material is important to understand how a material exhibiting viscoelastic properties performs.

As used herein, the term "hyaluronic acid" (hyaluronan or hyaluronan acid) refers to a polymer of disaccharide units consisting of glucosamine and glucuronic acid [1-3] linked by β 1-4, β 1-3 bonds and salts thereof the term hyaluronic acid thus refers to both the natural and synthetic forms of hyaluronic acid, a water-soluble polysaccharide of naturally occurring Hyaluronic Acid (HA), disaccharide units comprising D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), which are alternately linked to form a linear polymer, a high molecular weight HA may comprise 100 to 10,000 disaccharide units.

Other glycosaminoglycans (GAGs) may also be used in the hydrogel. These glycosaminoglycans include Chondroitin Sulfate (CS) and Dermatan Sulfate (DS) forms, polymers of glucuronic acid and galactosamine, and polymers of Heparan Sulfate (HS) and Heparin (HP), glucuronic acid and glucosamine. The degree and pattern of sulfation of these GAGs is of critical importance, as the sulfation pattern determines the formation of complexes with multiple families of proteins (e.g., coagulation proteins, growth factors, cytokines, neutrophils). See, e.g., Powell AK, Yates EA, Fernig DG, Turnbull JE. interactions of heparin/heparin sulfate with proteins, aprazal of structural factors and Experimental protocols, Glycobiology, 2004, month 4; 14(4):17R-30R ] those suitable for optimizing implanted patch implants include hyaluronic acid, non-sulfated GAGs and minimally sulfated GAGs (such as the Chondroitin sulfate form present in the stem cell niche), such as Karumbaiah L et al, Chondrotin Sulfate Glycoamino glyco-polysaccharides Hydrogels Create endogenesis Niches for Neural Stem cells bioconjugate Chem.2015.12, 16; 26(12) 2336-49 and Hayes AJ et al, Chondroitin sulfate as reactive biologizers for isolation of insulating porous promoter cells.J. Histochem Cytochem.2008, 2 months; 56(2) 125-38 (incorporated herein by reference).

As used herein, the term "cell" refers to one or more cells in an implant. The cells of the present disclosure are eukaryotic cells. In some embodiments, the cell is of animal origin, optionally from a human organ, and may be a stem cell, a mature somatic cell, a progenitor cell, or an intermediate cell in the lineage stage from a stem cell to a mature cell. The term "cell population" or "cell" refers to a combination of one or more cells of the same or different origin, of the same or different cell types; the term is used interchangeably herein with the term "donor cell," which means a cell that may be autologous or allogeneic. In some embodiments, the cell population may be derived from a cell line, from freshly isolated cells, or in some embodiments, the cell population may be derived from a portion of an organ or tissue, optionally from a donor or recipient.

The term "stem cell" refers to a population of cells that can self-replicate (produce the same daughter cells as the mother cells) and have the ability to differentiate multiple times (i.e., can produce more than one type of adult cell). As used herein, the term "progenitor cell" or "precursor" is defined broadly to encompass the progeny of a stem cell and its progeny. Progenitor cells are populations of cells that may have multi-, bi-or mono-differentiation properties, but minimal, if any, self-replicating capacity. Committed progenitors are progenitors that have unidifferentiation properties and can differentiate into specific lineages, resulting in only one mature cell type. Non-limiting examples of stem cells include, but are not limited to, Embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, germ layer stem cells, defined stem cells (ectodermal, mesodermal or endodermal), perinatal stem cells, amniotic fluid derived stem cells, Mesenchymal Stem Cells (MSCs), hemangioblasts, and those derived from umbilical cord, Wharton's glue and/or placenta. Intermediate cells between stem cells and committed progenitors include populations of cells such as hepatoblasts and pancreatic duct progenitors, as well as other forms of transient proliferative cells that may have multi-differentiation potential, but broad proliferative potential, but more limited, if any, self-replicating capacity.

The term "mesenchymal cells" refers to cells derived from the mesenchyme, including but not limited to angioblasts, precursors of endothelium, precursors of stellate cells, endothelium, stellate cells, stromal cells, various subpopulations of mature and progenitor cells, and Mesenchymal Stem Cells (MSCs), which are multipotential stromal cells, and various subpopulations of mature and progenitor mesenchymal cells. MSCs are populations of Cells prepared from tissues by culture selection processes (Cathey et al Stem Cells 2018; PMID: 29732653; Graceb et al Biochimie2018: PMID 29698670; Caplan AI. Stem Cells int.2015; PMID: 26273305. mature mesenchymal Cells are of at least two general classes, (a) mature mesenchymal Cells (astrocytes/stromal Cells) that produce and are surrounded by extracellular matrix forms comprising fibrillar collagen (e.g., type I collagen, type III collagen, type V collagen) and related matrix components (fibronectin, chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans) and binding signals that form complexes (e.g., growth factors, cytokines) and binding signals that form complexes with Cells of the generally linear (stringy) cell population Tendon, stroma, and myofibroblasts. (b) Mature mesenchymal cells, such as endothelium, produce and are surrounded by extracellular matrix forms comprising network-like collagens (e.g., type IV collagen, type VI collagen, type VIII collagen, type X collagen) and associated matrix molecules (fibronectin, heparan sulfate proteoglycans, heparin proteoglycans) and binding signals (e.g., growth factors, cytokines) that are collectively associated with cells with more squamous or cubic or cobblestone morphology. Non-limiting examples of such cells include endothelium and myoepithelium.

Precursors of these mesenchymal cell types include, but are not limited to, angioblasts, which have pluripotency and can differentiate into endothelial lineages (their advanced stages are fenestrated endothelium) or stellate cells (their advanced stages are myofibroblasts (stroma). MSCs can optionally be prepared by culture selection methods (Cathy et al Stem Cells 2018; PMID: 29732653; Graceb et al Biochimie2018: PMID 29698670; Caplan AI. Stem Cells int.2015; PMID: 26273305).

The term "epithelial cell expansion" is associated with the diameter of epithelial cell colonies that typically form colonies of cubic or cobblestone morphology, and estimates of growth are associated with complexes of the diameters of the cells of the colonies. In contrast, estimates of growth of mesenchymal cell colonies correlate with the density of the colonies, as mesenchymal cells are more mobile and mobile, and colony density is a reflection of the net total number of cells remaining within the colony boundary.

The term "epithelial cells" refers to cells derived from the epithelium, specialized cells that provide multiple functions required for the tissue and/or the whole body of the host. They are believed to have the ability to migrate as precursors or immature cells; as they mature, they become quiescent and form squamous or cobblestone-like or columnar polarized cells with apical, basal and lateral faces, and bind to each other through various junctions (connexins, tight junctions, adherence). Their amplification potential is expressed in the diameter of the colony (rather than in density). Mature epithelial cells provide a variety of functions, such as specialized product secretion or contributing to metabolism (hepatocytes, cholangiocytes), detoxification (hepatocytes), enzyme production (acinar cells), endocrine factor production (e.g., pancreatic islets or other endocrine cells), electrical activity (neuronal cells), and absorption (intestinal cells).

The term "biliary stem cell" (BTSC) refers to an epithelial stem cell present throughout the biliary system and located extramurally and intramurally as well as intracryptic Pericholecystal (PBG), bulonna glands of the gallbladder villus. They have the ability to convert to committed liver and/or pancreatic progenitors. Liver offspring enter hepatic sinusoids via herring's canal; pancreatic progenitor cells are present within the Pancreatic Ductal Gland (PDG), localized to regions of the biliary system within the pancreas.

To date, at least 7 subpopulations of stem cell populations have been identified by overlapping traits, ranging from extremely primitive BTSCs to stem cell populations that can be defined as liver stem cells or pancreatic stem cells. A description of these known matters is given below. The most primitive cells are found in both extramural peribiliary glands-cells tethered to the surface of the bile duct-and intramural peribiliary glands-cells found within the wall of the bile duct. The intramural pericholecystal glands (PBGs) near the fibromuscular layer in the center of the bile duct wall can also be considered as crypts (parallel to the intestinal crypts), where niches for the most primitive stem cell population are present. A large number of PBGs within the biliary network are present within the hepatopancreatic common duct and within the large intrahepatic bile ducts. PBG is not present in the gallbladder, and the stem cell niche within the gallbladder is the bottom of the gallbladder villi containing a metaphase to late stem cell population (precursors of liver stem cells). BTSC are precursors to both the liver and pancreas. They produce liver stem cells (precursors of the liver) and pancreatic stem cells (precursors of the pancreas), and these stem cells are present in the entire biliary system, but the number is affected by the proximity of the liver or of the pancreas. Thus, a small number of pancreatic stem cells and a large number of liver stem cells are localized in the PBGs of the large intrahepatic bile ducts, while a small number of liver stem cells and a large number of pancreatic stem cells are localized in the PBGs of the hepatopancreatic common duct.

A summary of genetic markers is provided in the figures, generally, all BTSC subpopulations express general biomarkers including endodermal transcription factors for both liver and pancreas (e.g., SOX9, SOX17, PDX1), pluripotency genes (e.g., OCT4, SOX 4, NANOG, SALL4, KLF4/KLF 4, BMI-1), one or more of the hyaluronic acid receptor isoforms (standard and/or variant isoforms) of CD4, CXCR4, and cytokeratin 8 and cytokeratin 18. the stem cell subpopulations within the biliary and PBG include (1) bunner gland stem cells in the submucosa of the duodenum, which express 4, TRA-160 and 181, and which have a trait that is distinguishable from stem cells in the intestinal tract, (2) LG intramural lineages (36SC) which express sodium iodide in the same direction as pancreatic reticulocytes (NIS) and are absent in 4, and are found in the bile duct epithelial stem cells of the liver, and have a high or a pancreatic endothelial cell adhesion to pancreatic epithelial cells (PBACH), and are found in the liver epithelial stem cells of the liver, and are found in the liver epithelial cells of the liver, and are found in the liver.

Notably, when the liver stem cells and pancreatic stem cells are in an early developmental stage (e.g., as ESCs or other cells), they may also be present in their respective organ of origin, and any of those cells disclosed herein may alternatively be produced by induction (i.e., as ipscs).

As used herein, the term "supportive" is used to describe cells that are capable of assisting cells to proliferate from another lineage stage or to assist neighboring cells by producing "paracrine signals," which are factors that are active in terms of survival, expansion, migration, differentiation, and maturation in terms of effects on neighboring cells. For example, supportive mesenchymal cells may be defined by their ability to affect epithelial cells, optionally by secretion of Matrix Metalloproteinases (MMPs) and/or one or more paracrine signals or growth factors. Many of these cells have been outlined in recent reviews. (Cathey et al Stem Cells 2018; PMID: 29732653; Graceb et al Biochimie2018: PMID 2969870; Caplan AI. Stem Cells int.2015; PMID: 26273305).

For hepatic stem cells or biliary stem cells, these partners consist of hemangioblasts (CD117+, CD133+, VEGFr +, CD31 negative) and their direct progeny, precursors of the endothelium (CD133+, VEGFr +, CD31+, wecker factor (vWF +)) and precursors of the stellate cells (CD146+, ICAM-1+, α -smooth muscle actin + (ASMA), vitamin a negative), which can be mimicked, in part and/or to some extent, by the use of Mesenchymal Stem Cells (MSCs), such as but not limited to stem cells derived from bone marrow or adipose tissue.

Intermediate cells in the lineage network are termed "transient proliferating cells," which are cells that can have bi-differentiation (or multi-differentiation) potential, have substantial proliferation potential but exhibit little, if any, true self-replication, have low to moderate (or even no) pluripotency gene expression, and express traits indicative of fate directed to the liver (e.g., albumin, alpha-fetoprotein) or pancreas (e.g., insulin, MUC6, amylase). These cells include hepatoblasts (the network produces the liver) and pancreatic duct progenitors (the network produces the pancreas).

As used herein, the term "pancreatic ductal progenitor cells" refers to the bilaterally competent cells present within the Pancreatic Ductal Gland (PDG) within the pancreas and producing acinar cells and islets in our study, we found that they express SOX9, PDX1, PTF1a, HNF1 β, EpCAM, LGR5, ICAM-1, CD44, and a subset that express NGN3 or MUC6 or amylase, which are also widely characterized in other studies, see, e.g., Rezanejad H, Ouziel-Yahalom L, KeyKezer CA, Sullivan BA, Hollie-Lock J, Li WC, Guo L, Deng S, Lei J, Markmann J, Bonner-Weir S.738 heterology of SOX9and HNF1 β. Cell pages 3. 13; 10. mu. 13. Rep.) (725.

As used herein, the term "hepatoblasts" refers to bilaterally competent liver cells that can give rise to the hepatocyte and cholangiocyte lineages and are present in PBGs in or near the canals of herring or within the large intrahepatic bile ducts, they have extraordinary proliferation (i.e., expansion) capacity, but very low, if any, self-replicating capacity relative to observations in hepatic stem cells or BTSCs, these cells are characterized by overlapping, but distinct from biomarker profiles of hepatic stem cells or biliary stem cells, they express SOX9, low (or even negligible) levels of SOX17, high levels of LGR5, HNF4- α, and EpCAM, which are predominantly present at the plasma membrane and express P450a7, cytokeratin 7, secretin receptors, stable expression of albumin in all hepatoblasts, high levels of formazan protein (AFP), intercellular adhesion molecule (ICAM-1) but not NCAM-1, and negligible or non-pluripotent gene expression (e.g. multiple gene expression of clon P735, KL 3, e.g. nank 3, KL 3, and no substantial marker such as nax 355631, KL.

As used herein, the term "committed progenitor" refers to a single differentiated competent progenitor cell that produces a single cell type (e.g., committed hepatocyte progenitor cells). In some embodiments, they do not express pluripotency genes. Committed hepatocyte progenitors are identified by the expression of albumin, AFP, glycogen, ICAM-1, various enzymes involved in glycogen synthesis, and gap junction genes (connexin 28). These cells produce hepatocytes. The committed biliary (or cholangioepithelial) progenitor cells give rise to biliary epithelial cells and are identified by the expression of EpCAM, cytokeratin 7 and cytokeratin 19, aquaporins, CFTR (cystic fibrosis transmembrane conductance regulator) and membrane pumps associated with bile production. In some embodiments, the committed islet progenitor cells express insulin, glucagon, and other islet hormones, albeit at very low levels; as it matures, the level of islet hormone expression increases, but certain cells preferentially express certain hormones.

As used herein, the term "aggregate" refers to a plurality of cells that are aggregated together. The size and shape of the aggregates may vary, or the size and/or shape may be substantially uniform. The cell aggregates used herein may have various shapes such as spheres, cylinders (preferably equal in height and diameter) or rods, and the like. In one embodiment of the present disclosure, it is generally preferred that the aggregates be spherical or cylindrical, although other shaped aggregates can be used. The term "non-aggregated" refers to single or unicellular stem and/or progenitor or mature cells. In some embodiments, the compositions provided herein can comprise substantially aggregated cells, substantially non-aggregated cells, or mixtures thereof.

The term "organoid" refers herein to a specific cell aggregate of donor epithelial cells and mesenchymal cells that self-assemble by the simple panning process described herein. In some embodiments, the mesenchymal cell is a supporting mesenchymal cell. In some embodiments, organoids are formed following culture on low-adherence culture dishes under defined serum-free conditions tailored for multiple lineage stages of aggregated cells in suspension. Other studies have also used specific matrix extracts (such as Matrigel) to prepare organoids. Indeed, this material is known to be an industry standard. See Hindley et al dev.biology 2016; 420:251-261. PMID 27364469. For the use of these organoids in the patch implants described in the present invention, the described conditions for maintaining these organoids would not be successful. Factors such as those present in Matrigel will prevent or substantially reduce MMP production by the cells required for success of these patch implants. Furthermore, Matrigel cannot be used as a component of conditions under which cells are clinically used for human or veterinary purposes.

The term "culture" or "cell culture" means the maintenance of cells in an artificial in vitro environment. "cell culture system" is used herein to refer to culture conditions under which a population of cells can be grown ex vivo (outside the body).

"Medium" is used herein to refer to a nutrient solution used for cell culture, growth, or proliferation. The medium may be characterized by functional properties such as: such as, but not limited to, the ability to maintain the cells in a particular state (e.g., pluripotent, proliferative, resting, etc.) to mature cells-in some cases, specifically, the ability to promote differentiation of progenitor cells into cells of a particular lineage. A non-limiting example of a culture medium is Serum Supplemented Medium (SSM), which is any basal medium supplemented with a certain level (typically about 10% to about 20%) of serum. The serum may be autologous (same species as the cells), or more commonly serum from animals that are routinely slaughtered for commercial purposes (e.g., chickens, cows, pigs, etc.). Notably, embodiments of the invention directed to stem cells employ media that avoid binding to serum and/or serum components that drive differentiation. The depot column medium is a serum-free medium designed for endodermal stem/progenitor cells, comprising a basal medium (nutrients, amino acids, vitamins, salts, carbohydrates), free of copper, containing a small amount (<0.5mM) of calcium, supplemented with selenium, zinc, insulin, transferrin, lipids, but free of cytokines or growth factors. Other media that support stem cells have been found to be useful, but they must avoid any factor that causes cell differentiation, as the maturation process will result in silencing of membrane-associated and/or secreted MMP production.

The basal medium is a buffer for cell culture and is composed of various nutrients in a composition of amino acids, sugars, lipids, vitamins, minerals, salts, trace elements, and chemical components that mimic the interstitial fluid around the cells. Furthermore, the cell culture medium is usually composed of a basal medium supplemented with a small amount (usually 2% -10%) of serum. For the implantation techniques described herein, the conditions are used to maintain the cells as stem cells or early progenitor cells, so serum or any typical supplement that might drive the cells toward mature cell fate should be avoided. In addition to conventional basal media, there are various nutritional supplements, lipids (a mixture of free fatty acids complexed with albumin and carrier molecules such as high density lipoproteins). Only two hormones/growth factors were added: insulin (required for carbohydrate metabolism) and transferrin (required as the Fe carrier for polymerase). The depot column medium is a serum-free medium designed for endodermal stem/progenitor cells comprising a basal medium, containing no copper, a small amount (<0.5mM) of calcium, supplemented with zinc, selenium, insulin, transferrin, lipids, but no cytokines or growth factors. The use of other growth factors and cytokines, especially serum, should be avoided as they induce donor cell differentiation, thereby minimizing the production of MMPs required for implantation and migration processes.

As used herein, "depot column medium" refers to any medium that is free of copper, calcium (<0.5mM), selenium, zinc, insulin, transferrin/Fe, a mixture of free fatty acids bound to purified albumin, and optionally also contains High Density Lipoprotein (HDL). In some embodiments, the depot tower medium comprises any medium (e.g., RPMI1640 or DMEM-F12) that is copper-free, contains a small amount of calcium (e.g., 0.3mM),. about.10-9M selenium,. about.0.1% bovine serum albumin or human serum albumin (highly purified and free of fatty acids),. about.4.5 mM nicotinamide,. about.0.1 nM zinc sulfate heptahydrate,. about.10-8M hydrocortisone (an optional component for liver precursors but not pancreas precursors),. about.5 μ g/ml transferrin/Fe,. about.5 μ g/ml insulin,. about.10 μ g/ml high density lipoprotein, and a mixture of purified free fatty acids (added after the free fatty acids are bound to the purified serum albumin). The free fatty acid mixture consists of-100 mM of each of palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids. Non-limiting exemplary methods for preparing the media have been disclosed elsewhere, for example, Kubota H, Reid LM, proc.nat.acad.scienn. (USA) 2000; 97: 12132-.

Thus, in some embodiments, the conditions of these patch implants are reversed from conventional use of medium supplemented with small amounts (typically 2% -10%) of serum. Serum has long been added to provide the necessary signaling molecules (hormones, growth factors, cytokines) required to drive biological processes (e.g., proliferation, differentiation). In some embodiments, serum is not included to avoid differentiation of cells and/or to avoid inactivation or silencing of MMP production (particularly in secreted form).

As used herein, the term "effective amount" (or effective amount) refers to an amount sufficient to treat a disease state or condition (e.g., liver disease or pancreatic disease). An effective amount may be administered in one or more administrations, applications or doses. Such delivery depends on many variables, including the time period over which the individual dosage units are used, the bioavailability of the composition, the route of administration, and the like. It will be understood, however, that the specific amount of the composition for any particular patient will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex and diet of the patient, the time of administration, the rate of excretion, the composition combination, the severity of the particular disease being treated (e.g., liver or pancreatic disease), and the mode of administration.

The terms "equivalent" or "biological equivalent" when referring to a particular molecule, biological material, or cellular material are used interchangeably and refer to those substances having minimal homology while still maintaining the desired structure or function.

As used herein, the term "expression" refers to the process of transcription of a polynucleotide into mRNA and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein, and if the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample; in addition, the expression levels of multiple genes can be determined to establish an expression profile for a particular sample.

As used herein, the term "function" may be used to modify any molecule, biological material, or cellular material to achieve a particular specified effect.

As used herein, the term "gene" is meant to broadly include any nucleic acid sequence that is transcribed into an RNA molecule, whether the RNA is coding (e.g., mRNA) or non-coding (e.g., ncRNA).

As used herein, the term "produce" and equivalents thereof (e.g., generating, generated, etc.) are used interchangeably when referring to method steps for producing organoids of the present disclosure.

As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials.

The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. The polynucleotide may have any three-dimensional (3D) structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, RNAi, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA having any sequence, isolated RNA having any sequence, nucleic acid probe, and primer.

Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise indicated or required, any aspect of the technology (i.e., polynucleotide technology) encompasses the double-stranded form as well as each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer in the broadest sense to a compound that is an amino acid, amino acid analog, or peptidomimetic of two or more subunits. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other linkages (e.g., ester, ether, etc.). The protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can constitute the sequence of the protein or peptide. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine as well as both D and L optical isomers, amino acid analogs, and peptidomimetics.

As used herein, the terms "subject" and "patient" are used interchangeably and are intended to mean any animal. In some embodiments, the subject may be a mammal. In some embodiments, the mammal is a bovine, equine, porcine, canine, feline, simian, murine, human, or rat. In some embodiments, the subject is a human.

The term "tissue" is used herein to refer to the tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. The tissue may be healthy, diseased, wounded, damaged, and/or have a genetic mutation. As used herein, the term "native tissue" or "biological tissue" and variations thereof refer to biological tissue that, when derived from an organism, exists in a native state or an unmodified state. "micro-organ" refers to a portion of a "bioengineered tissue" that mimics a "native tissue".

A biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues that constitute an organ or portion or region of an organism. The tissue may comprise a homogeneous cellular material, or it may be a composite structure, such as that present in a region of the body comprising the breast, which may comprise, for example, lung tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to, those derived from liver, pancreas, biliary system, lung, intestine, thyroid, thymus, bladder, kidney, prostate, uterus, breast, skin and subcutaneous dermal tissues, brain, spinal cord, blood vessels (e.g., aorta, iliac veins), heart, muscle, including any combination thereof.

As used herein, "treating" (or treating) a disease in a subject refers to (1) preventing the appearance of symptoms or disease in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results can include, but are not limited to, one or more of alleviation or amelioration of one or more symptoms, diminishment of extent of a disorder (including disease), stabilization of state of a disorder (including disease) (i.e., not worsening), delay or slowing of a disorder (including disease), progression, amelioration or palliation of a disorder (including disease), state, and detectable or undetectable remission (partial or complete).

Abbreviations

AFP, alpha-fetoprotein; ALB, albumin; BTSC, biliary stem cells; CD, common determinant; CD44, hyaluronic acid receptor; CD133, prominin; CFTR, cystic fibrosis transmembrane conductance regulator; CK, cytokeratin; CXCR4, CXC-chemokine receptor 4 (also known as fusion viral protein or CD 184; also known as platelet factor 4); EGF, epidermal growth factor; ELSMC, early lineage mesenchymal cells, consisting of angioblasts and their progeny, precursors of the endothelium and precursors of the stellate cells; EpCAM, epithelial cell adhesion molecule; FGF, fibroblast growth factor; HB, hepatoblasts; HGF, hepatocyte growth factor; HpSC, liver stem cells; KM, depot column medium, a serum-free medium designed for endoderm stem cells; KRT, cytokeratin gene; LGR5, G protein-coupled receptor 5 containing leucine-rich repeats that binds to R-spondin; MMPs, matrix metalloproteinases, a large family of proteases associated with the lysis, cell migration and regeneration reactions of the extracellular matrix; NANOG, a transcription factor critically involved in self-renewal; NCAM, neural cell adhesion molecule; NIS, sodium/iodine symporter; OCT4, (octamer-binding transcription factor 4) also known as POU5F1(POU domain, class 5, transcription factor 1), a gene expressed by stem cells; PDX1, pancreatic and duodenal homeobox 1, transcription factors critical to pancreatic development; PBG, pericholecystal gland, stem cell niche for biliary stem cells; SALL4, Sal-like protein 4, was found to be important for the autonomous replication of stem cells; SOX, Sry related HMG box; SOX2, a transcription factor important for maintaining self-renewal, or pluripotency of embryonic stem cells and defined stem cells. SOX9, a transcription factor associated with endosymous tissues (liver, intestine and pancreas); SOX17, a transcription factor important for liver differentiation; VEGF, vascular endothelial growth factor; vWF, von willebrand factor.

Embodiments of the present disclosure

In the examples provided herein, applicants have created patch implants, a novel method for transplanting cells into an internal organ, the design characteristics of which depend on whether the cells are stem or mature cells. Applicants demonstrate herein these methods wherein an implant of biliary stem cells (BTSCs), precursors of liver and pancreas, is transplanted into the liver or pancreas. The host used to develop these methods is a breed of domestic pigs (Sus scrofa therapeutics). They are the major animal species used for transformation studies, surgical models and procedural training, and are increasingly used as a replacement for monkeys in preclinical studies.

Organoids containing organoids are placed on a Seri-silk substrate (mesh material), impregnated on the plasma membrane side of the Seri-silk substrate with a thicker HA hydrogel (-700 Pa) that is surgically or tethered to the surface of the liver or pancreas, the implant causes remodeling of the organ capsule and adjacent tissues, and optionally distant parenchymal tissue, followed by merging of donor cells and host cells, by two weeks the donor cells have matured to a functional adult fate, such as hepatocytes (albumin) or islets of langerhans (β cellulin), by three weeks the histology of the organ capsule and normal tissues is restored.

These results of these examples are in contrast to past work on cell transplantation from solid organs to internal organs, where transplantation was achieved by direct injection or by cell delivery via a vascular route (see reviews by Bhatia et al, Lanzoni et al, Weber, etc.). Past transplantation methods have produced small numbers of cells to be implanted, with a potentially life-threatening risk of embolism, and significant levels of ectopic cell distribution. These problems can result in minimal or incomplete use of cell therapies directed to internal solid organs.

The patch implant strategy provides an alternative approach to cell therapy that is capable of delivering a sufficient number of cells and integrating them into the tissue, thereby significantly restoring one or more functions. These examples demonstrate safety provided that the biomaterials and substrate support used maintain the immature state of some or all of the donor cells, so a relevant pool of MMPs can be generated. A common cause of failure is any factor that causes donor cell differentiation. Without being bound by theory, it is contemplated herein that purified MMPs can be incorporated into implant biomaterials and/or that cells can be transformed using recombinant expression systems or other genetic modification techniques to secrete MMPs as an alternative to providing cells in a naturally occurring essential MMP implant. In such embodiments, the combination of MMPs bound to or transduced via the construct should include those identified in the expression profile provided in the examples below.

Composition of patch implant

Aspects disclosed herein relate to patch implants comprising a layer comprising a single cell population or two or more cell populations (e.g., donor cells that may be autologous or allogeneic) and a source of MMPs and a substrate comprising a biocompatible, biodegradable material that can be used to tether the implant to a target site. In some embodiments, the one or more cell populations comprise a population of epithelial cells and a population of mesenchymal cells. In some embodiments, the cell population must be maintained in a particular state or "lineage stage" as part of the implant, meaning that they do not differentiate or mature further until incorporation into the organ. This can be achieved by maintaining a balance of variables relating to cell origin, MMP content, culture medium used and substrate quality. Each of these aspects is described in more detail below.

Without being bound by theory, it is believed that patch implants with (1) an optimal population or mixture of cells in culture medium and hydrogel-such as donor epithelial cells and a supporting mesenchymal stem/progenitor cell population that produces membrane-associated and/or secreted MMPs-that do not result in differentiation of the supporting mesenchymal stem/progenitor cell population or contain the appropriate MMPs, and (2) a substrate suitable for tethering the implant to a target site and preventing migration of cells in the implant towards the substrate, away from the target site, can be achieved successfully.

Exemplary cells

Without being bound by theory, the cells may be of any mature lineage stage, including Embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, defined stem cells, committed progenitor cells, transient proliferating cells, or mature cells. However, in certain embodiments, the source of MMPs must be present in the patch implant. Thus, a cellular source of MMPs for patch implants is contemplated herein. Such cell sources must be in an early lineage stage capable of expressing membrane-associated and/or secreted matrix metalloproteinases. A non-limiting example of such an early lineage stage is early lineage stage mesenchymal stem cells (ESMLC).

In some embodiments, the cells to be implanted are epithelial cells collocated with mesenchymal cells. In some embodiments, the epithelial cells comprise epithelial stem cells. In some embodiments, the epithelial cells comprise committed and/or mature epithelial cells. In some embodiments, the committed and/or mature epithelial cells comprise mature parenchymal cells. In some embodiments, the mature parenchymal cells include one or more of hepatocytes, cholangiocytes, or islet cells. In some embodiments, the mesenchymal cells comprise ELSMC. In some embodiments, the ELSMC includes one or more of angioblasts, precursors of endothelium, precursors of stellate cells, and MSCs. In some embodiments, the epithelial cells and mesenchymal cells are lineage-collocated with each other. In some embodiments, the epithelial cells and mesenchymal cells are not lineage stage partners of each other, e.g., are not at about the same lineage stage or maturation stage, respectively. In some embodiments, the epithelial cell is a mature cell. In some embodiments, the mesenchymal cells are ELSMCs.

In some embodiments, at least one of the epithelial cells and the mesenchymal cells are derived from a donor. In some embodiments, the donor is a subject in need of tissue transplantation. In some embodiments, the donor is a source of healthy cells for tissue transplantation. In some embodiments, at least one of the epithelial cells and the mesenchymal cells is autologous to the intended recipient of the patch implant. In some embodiments, all cells (i.e., epithelial and mesenchymal cells) are autologous to the intended recipient of the implant. In some embodiments, the donor of the cells may be a donor other than the recipient (allogeneic), or may also be a subject with an internal organ that is diseased or dysfunctional (autologous), optionally wherein the donor is obtained from a portion of the internal organ that is not diseased or dysfunctional and/or has been genetically modified to restore function to the cells.

In another aspect, the mesenchymal cells are a lineage stage partner of the donor cell, e.g., at a comparable or corresponding lineage stage. In another aspect, the mesenchymal cells are not a suitable partner for the lineage stage of the donor cell. The mesenchymal lineage stage cells can be angioblasts, early lineage stage precursors of endothelial and/or stellate cells, mesenchymal stem cells, endothelial or stellate cells or derivatives of these cell populations.

For stem cell transplantation, epithelial cells should be paired with natural lineage-partner mesenchymal cells (precursors of angioblasts and/or endothelial or stellate cells). For adult epithelial cells, suitable partners include early lineage mesenchymal cells (ELSMCs) consisting of precursors of angioblasts and/or astrocytes and endothelial cells. Applicants have shown that implantation can be achieved using preparations of Mesenchymal Stem Cells (MSCs) in combination with adult cells. In some embodiments, certain MSCs may be preferred over other MSCs. Without being bound by theory, it is believed that the implant can be optimized by selecting a cell combination that requires minimal, if any, cell culture and that will avoid serum and matrix components that can drive cell differentiation. Without being bound by theory, it is also understood that the epithelial-mesenchymal relationship is important because paracrine signaling supports MMP production. However, mature epithelial cells that are collocated with mature endothelium will survive the implant and will be functional cells, but cannot be implanted. Thus, if mature epithelial cells are paired with a mature matrix to form an implant, the resulting implant may be fibrotic.

For treatment of diseased or dysfunctional organs, the cells may be from a donor other than the recipient (allograft), or may also be autograft, and so from a subject with a diseased or dysfunctional condition internal organ, optionally wherein obtained from a portion of the internal organ that has not been diseased or dysfunctional and/or to which the cells have been genetically modified to restore function.

For establishing a model system for studying disease, the cells may be cells that have the disease and are transplanted onto/into normal tissue in an experimental host.

In some embodiments, the epithelial cells may be stem cells combined with supporting mesenchymal cells (optionally ELSMCs) to form organoids (optionally self-assembled). These organoids may be embedded or contained in the hyaluronic acid hydrogel. The stem cells and/or progenitor cells of the present disclosure can include any stem cells and/or progenitor cells known in the art, including, for example, Embryonic Stem Cells (ESCs), Embryonic Germ Cells (EGCs), induced pluripotent stem cells (ipscs), Pancreatic Stem Cells (PSCs), hepatic stem cells (hpscs), biliary stem cells (BTSCs), hepatoblasts, pancreatic ductal progenitors, committed pancreatic progenitors, or committed hepatic progenitors. In some embodiments, the cell population includes only stem cells, such as pancreatic stem cells, liver stem cells, biliary stem cells (BTSCs), or buna-na stem cells. In other embodiments, the cells comprise only a subset of the multi-differentiation competent progenitor cells, such as hepatoblasts or pancreatic duct progenitor cells, or the implant may contain committed mono-differentiation competent progenitor cells (e.g., hepatocytes or biliary or pancreatic islet or acinar committed progenitor cells). In other embodiments, the cells comprise a mixture of stem cells and progenitor cells.

If adult epithelial cells are used, they can be mixed with ELSMCs in relevant ratios into the implant biomaterial. The ratio of the cell mixture can be determined to mimic the target tissue. Alternatively or additionally, the ratio may be determined by self-assembly of organoids. Organoids or cell mixtures are embedded in soft implant biomaterials (such as soft hyaluronic acid hydrogels). If stem cells are implanted, the stem cells and/or progenitor cells of the present disclosure can include any stem cells and/or progenitor cells known in the art, including, for example, Embryonic Stem Cells (ESCs), Embryonic Germ Cells (EGCs), Induced Pluripotent Stem Cells (iPSCs), Blanca stem cells (BGSCs), biliary stem cells (BTSCs), Pancreatic Stem Cells (PSCs), hepatic stem cells (HpSCs), transient proliferative cells (e.g., hepatoblasts or pancreatic duct progenitors), and committed mono-differentiated functional progenitors (e.g., committed pancreatic progenitors or hepatocyte progenitors or biliary epithelial progenitors). In some embodiments, the population of cells comprises only stem cells. In other embodiments, the cells comprise only a subpopulation of progenitor cells. In other embodiments, the cells comprise a mixture of stem cells and progenitor cells or a mixture of stem/progenitor cells and more mature cells. In yet other embodiments, a chimeric mixture of adult cells (e.g., hepatocytes, biliary epithelial cells, intestinal epithelial cells, pancreatic islets) and ELSMCs may be present.

Non-limiting examples include the use of a combination of assays that define self-replicating capacity and assays that demonstrate multi-differentiation capacity by morphological analysis, by gene and/or protein expression, cell surface markers, and the like, in some embodiments, stem cells and/or progenitor cells express at least one marker indicative of early liver cell lineage cells (e.g., SOX17, HNF-4 α, HNF6, HES1, CK19) and at least one marker indicative of early pancreatic cell lineage (e.g., PDX1, PROX1, NGN3, HNF β) for example, various combinations of pluripotent stem cell expression, such as SOX 4, clonx 4642, noc 364642, and SOX/or KLF 4642 may be identified by SOX9, SOX17, PDX1, CD133, NCAM, sonic hedgehog factor (sonic hedgehog) (SHH), sodium iodine symporter (NIS), nrr 8, CD 6866, OCT 44, and various combinations of these.

Thus, they will express one or more markers common to both the liver and pancreatic lineages (e.g., SOX9, LGR5/LGR6, EpCAM, CD133, CK19) and one or more markers of the early pancreatic lineage (e.g., SOX1, PROX1, NGN3, HNF β 1) for example, one or more markers of the early pancreatic lineage (e.g., PDX1, PROX1, NGN3, HNF β 1) may be identified by SOX9, SOX17, PDX1, CD133, NCAM, sonic hedgehog factor (sonic hedgehog) (SHH), sodium iodine symporter (lgs), LGR5, nir 6, and various pluripotent genes (e.g., OCT4, SALL 5845, nang) for example, klx 4624, kl 2, or a combination thereof.

Generation of mature cell types

The stem and/or progenitor cells can also differentiate into more mature cell types, if desired. This can be done in vitro by spontaneous differentiation and/or by direct differentiation. Direct differentiation may involve genetically modifying stem and/or progenitor cells to express a gene of interest or a combination thereof using defined media.

Non-limiting examples of defined media for differentiating cells include hormone-defined media (HDM) for differentiating endoderm stem cells into adult fates. Supplements can be added to the depot tower medium to produce serum-free, hormone-defined media (HDM) that facilitate the differentiation of normal hepatic or biliary stem cells to a specific adult fate. These supplements include calcium supplement to achieve a concentration equal to or greater than 0.6mM, 1nM triiodothyronine(T3)、10^-12M copper, 10nM hydrocortisone and 20ng/ml basic fibroblast growth factor (bFGF). The other medium conditions than those required for the selective production of hepatocytes (HDM-H) and cholangioepithelial cells (HDM-C) and pancreatic islets (HDM-P) were:

1) HDM-H: further supplemented with 7 μ g/L glucagon, 2g/L galactose, 10ng/ml Epidermal Growth Factor (EGF) and 20ng/ml Hepatocyte Growth Factor (HGF);

2) HDM-C: further supplemented with 20ng/ml Vascular Endothelial Growth Factor (VEGF) and 10ng/ml HGF; and

3) HDM-P: prepared without corticosteroids and further supplemented with 1% B27, 0.1mM ascorbic acid, 0.25 μ M cyclopamine, 1 μ M retinoic acid, 20ng/ml FGF-7 for 4 days, then replaced with the following supplements: 50ng/ml exenatide-4 and 20ng/ml HGF, induced for another 6 days.

The HDMs provided herein can be supplemented with additional growth factors, including, but not limited to, Wnt signaling, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), insulin-like growth factor (IGF), Transforming Growth Factor (TGF), Nerve Growth Factor (NGF), neurotrophic factors, various interleukins, Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor (PDGF), Stem Cell Factor (SCF), Colony Stimulating Factor (CSF), GM-CSF, erythropoietin, thrombopoietin, heparin-binding growth factor, IGF binding protein, and/or placental growth factor.

The HDMs provided herein can be supplemented with cytokines including, but not limited to, interleukins, lymphokines, monokines, colony stimulating factors, chemokines, interferons, and Tumor Necrosis Factor (TNF).

Applicants have shown that hyaluronic acid can affect stem and/or progenitor cells to express factors of key cell adhesion molecules required to modulate cell anchorage and cell-cell interactions, and to prevent internalization of those anchorage factors by stem and/or progenitor cells following cell suspension preparation, cryopreservation or transplantation non-limiting examples of such anchorage factors include integrins, which are large families of heterodimeric transmembrane glycoproteins that function to attach cells to extracellular matrix proteins of basement membranes, ligands on other cells, and soluble ligands-integrins contain large and small subunits, referred to as α and β, respectively-such subunits form β 0 αβ heterodimers, and at least 18 β and eight αβ subunits are known in humans, can produce 24 heterodimer integrations-in some embodiments, stem and/or progenitor cells express high levels of integrin subunits, such as ITG β, ITG β, ITG 3642, ITG 3653, ITG 3664, ITG B, ITG β, ITG 72, B, ITG β, and the like in embodiments of the cell ITG 3, itp 3, and B, ITG β, ITG 3.

In some embodiments, the stem cells and/or progenitor cells of the present disclosure differ from naturally occurring stem cells and/or progenitor cells at least in that they express the integrin subunit in an amount that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200% more than the amount of integrin subunit in the unmodified stem cells and/or progenitor cells. It is contemplated that an increase in integrin subunits can facilitate attachment of stem cells and/or progenitor cells to form cell-cell interactions, and prevent internalization of stem cells and/or progenitor cells if desired.

MMP

MMPs are one of the key factors that facilitate implantation and integration. MMPs consist of many isoforms (at least 28; in pigs, 24 isoforms are known), some are secreted (e.g., MMP1, MMP2, MMP7, MMP9), and others are plasma membrane associated (e.g., MMP14, MMP 15). Without being bound by theory, it is believed that a mixture of these isoforms is necessary for implantation, especially a mixture of secreted forms. All cells studied produced different amounts of secreted and membrane-associated forms, but stem/progenitor cells produced very high levels of secreted forms. Implantation depends on these secreted MMPs (membrane-associated forms have some known synergy). The cellular source of these isoforms is a practical way to provide the necessary MMPs to achieve implantation. As an alternative approach, applicants contemplate the genetic engineering of purified/recombinant forms of MMPs into implant biomaterials for binding to and/or cells in the implant to produce the necessary MMPs.

Cells can be successfully implanted so long as there is a source (ideally a cellular source) of a plurality of Matrix Metalloproteinases (MMPs), optionally in one or both of secreted and membrane-associated forms. MMPs are produced by all cell types (both immature and mature), but they differ in which isoform is produced and how the expression level of a particular MMP is. Representative secreted forms include MMP1, MMP2, MMP7, and MMP 9. Representative membrane-associated forms include MMP14 and MMP 15. It has been empirically found that the highest yield of secreted MMPs is produced by early lineage stage cells, stem cells and early progenitor cells. The biomaterial of the implant supports the ability of epithelial and mesenchymal cells to produce these various forms of Matrix Metalloproteinases (MMPs) that lyse the surrounding organ or tissue

And allows cells to migrate by solubilizing various forms of extracellular matrix components.

More generally, Matrix Metalloproteinases (MMPs) are a large family of zinc-dependent proteases that are involved in the breakdown and regulation of extracellular matrix components, and in transplantation, invasion, angiogenesis, vascularization and migration in both normal and pathogenic processes. There are at least 28 isoforms, including matrix protease, snake venom protease, astaxanthin, serratia protease, and the like. Their role in normal processes (such as transplants of the placenta) and pathogenic processes (such as invasion and metastasis of cancer) has been characterized.

The studies described herein provide evidence for novel effects of MMPs that contribute to the engraftment, migration and integration of transplanted cells. Stem/progenitor cells (both epithelial and mesenchymal) express multiple MMP isoforms that are particularly effective in these effects. Maturation of the cells results in silencing of the expression of one or more potent stem/progenitor-related MMPs, thereby reducing invasion and migration processes. Adult cells also express MMPs, mainly membrane-bound MMPs (MT-MMPs), which are involved in plastic processes, but not in the overall implantation and integration of cells into tissues. However, there is some synergy between MT-MMP and secreted forms. The net sum of this recognition is that, among other features, the implant biomaterial, substrate and other conditions must be one that optimizes the expression of various MMPs (such as secreted MMPs) so that implantation and migration processes can occur. Therefore, factors driving differentiation of transplanted cells will silence the complex MMP response in parallel. This recognition means that factors to be avoided include serum (which drives differentiation), soluble signals that drive differentiation (e.g., certain growth factors, cytokines, and hormones); extracellular matrix components that drive differentiation (e.g., collagen, adhesion molecules, highly sulfated glycosaminoglycans/proteoglycans); and mechanical forces (viscoelastic properties, which drive differentiation) that contribute to the consistency of the implant.

In some embodiments, the one or more cells in the mixture are a source of secreted and/or membrane-associated MMPs. Secreted MMPs may optionally be naturally produced by one or more of epithelial or mesenchymal cells, or optionally result from transformation of one or more of epithelial or mesenchymal cells with a recombinant expression vector or genetic editing for MMP production. In some embodiments, such as but not limited to embodiments involving a population of stem/progenitor cells that natively secrete MMPs, the variable that silences MMP expression-optionally membrane-associated and/or secreted MMP expression-is controlled in the patch implant. Non-limiting examples of such variables include variables that lead to stem/progenitor cell maturation such as, but not limited to, serum supplements to culture media or implant biomaterials, hormones or other soluble signals that affect differentiation of epithelial and/or mesenchymal cells, oxygen levels (since anaerobic conditions keep cells in an immature state, while higher oxygen levels promote differentiation), and the consistency of the implant material (since consistency or mechanical forces (such as shear forces) and compression can drive differentiation).

For stem cell implants, epithelial cells and their mesenchymal counterparts are optimally stem or progenitor cells, as both provide contributions from multiple types of MMPs. For implantation into adult cells, MMPs of epithelial or mesenchymal cells should optimally provide a cellular source of membrane-associated and/or secreted MMPs, e.g. optionally using ELSMC as a cellular source of membrane-associated and/or secreted MMPs. Thus, implants in which both epithelial and mesenchymal cells are mature cell types cannot be successfully implanted. Epithelial cells may survive and proliferate and function if they have mature endothelium, but cannot be implanted; if a mature matrix is present, fibrosis of the implant may occur.

In summary, implantation will occur if the epithelial-mesenchymal cell partner is a stem/progenitor cell, or if at least one of the epithelial or mesenchymal cells is a stem cell, for example optionally using ELSMC as a source of matrix-associated and/or secreted isoforms of Matrix Metalloproteinases (MMPs), or if purified/recombinant forms of those MMPs are provided in the implant biomaterial. The early lineage mesenchymal cells (ELSMCs) suitable for use in patch implants may be hemangioblasts, precursors of endothelium, early lineage endothelium, precursors of stellate cells, early stellate cells or Mesenchymal Stem Cells (MSCs) or mixtures thereof.

Thus, contemplated herein are compositions for use as patch implants comprising at least a population of cells (e.g., epithelial and mesenchymal cells) and a source of MMPs (i.e., a population of cells in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), optionally supported by the conditions of the culture medium and/or hydrogel).

Media Components

For use in combination with the sources of cells and MMPs disclosed herein, any medium (including nutrients, vitamins, salts, etc.) plus key soluble factors (such as insulin, transferrin/Fe and lipids, which are found to be useful for expansion and/or survival of stem/progenitor cells) can be used. All factors that lead to cell maturation must be avoided as maturation will lead to reduced or silenced MMP expression. Factors to be avoided include serum, soluble signals to drive differentiation, extracellular matrix components to drive differentiation, and consistency or mechanical forces (compression, abrasion). A non-limiting example of such a medium is a depot column medium.

Thus, contemplated herein are compositions for use as patch implants comprising at least a population of cells and a source of MMPs (e.g., a population of cells in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a suitable culture medium or purified MMPs). A non-limiting example of a suitable medium is a depot column medium. Other stem cell media, such as those used for Embryonic Stem (ES) cells or Induced Pluripotent Stem (iPS) cells, are equally suitable provided that they do not contain soluble or matrix signals that drive differentiation of the cells, are a source of MMPs, or are present or include MMPs from other sources.

Hydrogels

The patch implant comprises one or more hydrogel components. In some aspects, biomaterials that can form hydrogels or parallel insoluble complexes (e.g., non-collagenous gelatin) include hyaluronic acid, thiol-modified hyaluronic acid, other glycosaminoglycans (GAGs), or combinations thereof. The trigger for curing may be any factor that initiates crosslinking of the matrix components or gelling of those components where gelling may occur. The cross-linking agent may comprise a poly (ethylene glycol) (PEG) or PEG-diacrylate (PEGDA) hydrogel or disulfide bond-containing derivatives thereof. Notably, the biomaterial included in the hydrogel should be selected for its ability to support the dryness of the disclosed cell population or populations used in patch implants (e.g., ELSMCs).

Matrix components that support maintenance of dryness can be used, but components that drive differentiation cannot be used. Non-limiting examples of supportive components include hyaluronic acid or non-sulfated (or minimally sulfated) glycosaminoglycans. These components are particularly useful because they can be "tuned", i.e. modified, to have different levels of consistency (optionally measured as viscoelasticity). Thus, in some aspects, the cell population (optionally cells of an isolated internal organ) may be solidified ex vivo within the biological material prior to introducing the cells into the host, or in the alternative, injected with a fluid substance and allowed to solidify in vivo.

Very soft (e.g., -100 Pa) hydrogels are ideal for maintaining the immature state of the donor cells. A more dense (e.g., >500Pa) version can be used to mature cells enough to cut MMP production, thereby blocking migration. The thicker version may also minimize adhesion of adjacent tissues. In certain embodiments, the cell population and the source of MMPs, optionally another cell population (i.e., a cell population at an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs)).

Without being bound by theory, it is believed that the presence of the extracellular matrix form in the amniotic membrane can maintain the donor cells in an immature state. Thus, it is contemplated that amniotic membrane may be used for the hydrogel, and optionally as an alternative biocompatible, biodegradable material.

Notably, materials known to trigger maturation comprise certain components derived from the mature extracellular matrix, such as, but not limited to, type I collagen. These materials should be excluded from all elements of the patch implant, including but not limited to cells, hydrogels, culture media, substrates, and/or any additional components.

Thus, contemplated herein are compositions for patch implants comprising at least a population of cells and a source of MMPs (i.e., a population of cells in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a suitable medium and contained in a hydrogel).

As described above, consistency may drive the ability of cells to differentiate. Additional thick hydrogels may have an effect on cell migration ability. Since the cells have to migrate into the organ, the hydrogel comprising the cells should have sufficient viscoelastic properties to allow said cells to migrate optionally within or away from the hydrogel and/or patch implant. Non-limiting examples of such viscoelasticity include, but are not limited to, viscoelasticity in the range of about 50 to about 100Pa or about 250Pa, for example, at least about 50Pa, at least about 100Pa, at least about 150Pa, at least about 200Pa, at most about 250Pa, at most about 200Pa, at most about 150Pa, at most about 100Pa, and/or any single value therebetween, such as, but not limited to, about 50Pa, about 100Pa, about 150Pa, about 200Pa, and about 250 Pa.

Without being bound by theory, it is believed that as cells migrate from the patch implant to the target organ or tissue, they migrate with some of the hydrogel with which they are associated or with which they are coated. The hydrogel isolates the cells from signals in the tissue microenvironment that affect cell differentiation or maturation and maintain the cells in an immature state. This facilitates cell migration throughout the parenchymal tissue. As the hyaluronic acid in the hydrogel is gradually degraded and removed, the cells begin to differentiate or mature and turn on adult cell function.

Method of producing organoids

Without being bound by theory, it has been determined that early lineage cells can have a high success rate of implantation when incorporated into organoids or aggregates. Such tissue may optionally comprise early lineage stage epithelial and mesenchymal cells.

Accordingly, provided herein is a method of forming an organoid, the method comprising, consisting of, or consisting essentially of culturing a mixture of epithelial and mesenchymal cells in a vessel suitable for tissue culture and in the presence of a culture medium, removing adherent mature cells on the surface of the vessel by panning, and recovering a self-assembled organoid from a cell suspension in the culture medium. Also disclosed herein are compositions comprising the organoids so produced.

In some embodiments, the procedure involves panning to remove mature cells by allowing the cells to selectively, rapidly (15-30 minutes) adhere to a conventional culture dish under serum-free conditions at 37 ℃ because even under these conditions, mature cells express various matrix components that make the cells adhere. Multiple rounds (e.g., 4-5 rounds) of this panning process can enrich for early lineage stage cells in the cell suspension. The cell suspension was then transferred to a low-adherence culture dish, placed again in serum-free medium (designed for early lineage cells) and placed in an incubator overnight at 37 ℃. The conditions promote the self-assembly of lineage-matched epithelial and mesenchymal cells into organoids. Organoids can be obtained from a mixture of early epithelial (ES cells, iPS cells, defined stem cells, transient proliferative cells, progenitor cells) and early mesenchymal cells (angioblasts, precursors of endothelium, precursors of stellate cells).

Mixtures of adult epithelial cells and mature mesenchymal cells and chimeric mixtures of mature epithelial cells and early lineage mesenchymal cells (ELSMCs) do not typically produce organoids, but can be used as a mixture of cells in suspension in an implant biomaterial. If the mature epithelium (e.g., hepatocytes, cholangiocytes, islets, acinar cells, intestinal epithelial cells, etc.) is paired with mature mesenchymal cells (e.g., endothelium, stellate cells, stromal cells, myofibroblasts), the mixture will not trigger successful integration of the implant into the target site or organ, but will remain on the surface of the organ or tissue. If a chimeric mixture comprising somatic cells and stem/progenitor cells (e.g., mature hepatocytes and hemangioblasts) is used, implantation does not occur because there is a source of MMPs that allow for cell implantation and migration.

In another aspect, the cells of the isolated internal organ may be solidified ex vivo within the biomaterial prior to introducing the cells into the host, or in the alternative, injected with a fluid substance and allowed to solidify in vivo into the implant. Preferably, the cells are introduced at or near diseased or dysfunctional tissue and may be introduced via injection or implanted onto/into the tissue, or introduced using a suitable surgical method.

In another aspect, biomaterials that can form hydrogels or parallel insoluble complexes can include hyaluronic acid, thiol-modified hyaluronic acid, other glycosaminoglycans (GAGs). The trigger for curing may be any factor that initiates crosslinking of the matrix components or gelling of those components where gelling may occur. The cross-linking agent may comprise a poly (ethylene glycol) (PEG) or PEG-diacrylate (PEGDA) hydrogel or disulfide bond-containing derivatives thereof.

In another aspect, the present disclosure provides a method of forming an organoid by culturing a first type of cell (epithelial) with one or more second type of cell (mesenchymal), wherein the second type of cell is in a mature stage that is a suitable lineage partner for the first type of cell. In some embodiments, this may be achieved by: removing mature cells adherent to the culture dish by panning; transferring the nonadherent cells to a low-adherence culture dish and placing the nonadherent cells in a proper culture medium; and recovering the organoids self-assembled under these conditions. The first type of cell may be an epithelial stem cell, a committed progenitor of an epithelial cell, or a mature cell (e.g., a hepatocyte). The second type of cell may be a stem cell of mesenchymal lineage (e.g., hemangioblast, mesenchymal stem cell), a progenitor cell of these lineages (e.g., endothelial or astrocytic progenitor cell), or a mixture of mesenchymal cells during early lineage. It is essential that this formation does not occur under all conditions. For example, culturing in Matrigel does not produce organoids suitable for successful patch implantation. Although Matrigel-prepared organoids can be implanted, the extent of implantation will be reduced relative to organoids prepared under defined conditions. Furthermore, Matrigel cannot be used as a component of the conditions to be used in clinical products.

In another aspect, the present disclosure provides a method for implanting cells into an organ, the method comprising contacting a patch implant, the patch implant comprising a plurality of layers, the plurality of layers comprising a biocompatible, biodegradable substrate, the substrate being neutral in effect on donor cell differentiation; a second layer comprising one or more biological materials (such as hyaluronic acid) that can be cured, such as a hydrogel; a mixture of epithelial cells and supporting mesenchymal cells bound to the solidified biomaterial; and attaching the bandage-like structure to the target site by suture or surgical glue. Adding a layer of cured biomaterial to the serosal surface of the substrate, the cured biomaterial being prepared to achieve 400Pa or more at a level at least twice the level present in the soft biomaterial to which the donor cells are bound. Cells within the patch implant are able to implant and migrate into and throughout tissues/organs, which then mature into an associated adult fate depending on the microenvironment they are in. Higher pascal levels of biomaterial embedded or contained into the porous substrate can prevent cell migration in the wrong direction, and biomaterial added to the serosal surface of the implant can minimize cell adhesion to other organs and tissues.

Organoids

According to one embodiment disclosed herein, organoids, floating aggregates of biliary stem cells (hereinafter "BTSCs"), and mesenchymal cells during early lineage (hereinafter "ELMC") have proven to be the most successful methods of incorporating cells into implants. Disclosed herein are the following: BTSCs and ELMCs can be self-selected to be organoids by panning to remove mature star/stromal cells, and this has proven to be more efficient and effective in establishing appropriate epithelial-mesenchymal partners for the lineage stage of the implant. In another aspect, the present disclosure provides a method of forming an organoid by culturing a first type of cell with a second type of cell, wherein the second type of cell is a lineage-appropriate partner of the first type of cell, removing mature cells adherent to a culture dish by panning, and recovering the self-assembled organoid from the cultured suspension. The first type of cell may be an epithelial stem cell or a committed epithelial cell. The second type of cell may be a cell of mesenchymal lineage, a mesenchymal stem cell or a mesenchymal cell during early lineage. Other aspects relate to self-assembled organoids and uses thereof.

In some embodiments, the donor cell and/or the supporting mesenchymal cell express a matrix metalloproteinase (hereinafter referred to as MMP). Without being bound by theory, it is believed that MMPs allow for pooling of donor and host cells, as well as lysis of the gleason's capsule (or equivalent capsule around a tissue or organ). The disclosure herein provides the expression that, in some embodiments, early stem cells or ELMCs express high levels of MMPs, while mature hepatocytes express low levels of MMPs. In some embodiments, the collocation of mature hepatocytes with mature sinusoidal endothelium (CD31+ + +, VEGF receptor +, collagen IV + and CD117 negative) and those for adult biliary epithelial cells associated with mature stellate and stromal cells (ICAM-1+, ASMA +, vitamin a + +, collagen I +) results in cell aggregates that remain on the surface of the organ and are not effectively engrafted. In some embodiments, implantation of mature epithelial cells requires that they be paired with immature mesenchymal cells that produce MMPs necessary for implantation and migration.

According to one embodiment disclosed herein, floating aggregates of organoids, stem/progenitor cells (such as BTSCs and ELSMCs) are demonstrated to be the most successful cell presentation for patch implantation success. Disclosed herein are the following: BTSCs and ELSMCs can be self-selected to be organoids by: mature mesenchymal cells were removed by standard panning procedures against cells adherent to conventional culture dishes under serum-free conditions, and the remaining cells (those that were not adherent) were then cultured in low-adherence culture dishes and in serum-free defined medium. Organoids self-assemble under these conditions.

In another aspect, the present disclosure provides a method of forming an organoid by culturing a first type of cell (epithelial) with a second type of cell (mesenchymal), wherein the second type of cell is a lineage-appropriate partner of the first type of cell, removing mature cells adherent to a conventional culture dish by a panning procedure, and recovering the self-assembled organoid from a culture suspension on a low-adherent culture dish. The first type of cell may be an epithelial stem cell, a transient proliferative cell, a committed epithelial progenitor cell. The second type of cell may be a stem cell of a mesenchymal cell, a transient proliferative cell or a committed mesenchymal progenitor cell.

In some embodiments, the donor cell and/or the supporting mesenchymal cell express a matrix metalloproteinase (hereinafter referred to as MMP). Without being bound by theory, it is believed that MMPs cause lysis of the capsule around the tissue or organ and allow for pooling of donor and host cells. The disclosure herein provides the expression that, in some embodiments, early stem cells or ELMCs express high levels of MMPs, while mature hepatocytes express low levels of MMPs. In some embodiments, the collocation of mature hepatocytes with mature sinusoidal endothelium (CD31+ + +, VEGF receptor +, collagen IV + and CD117 negative) and those for adult biliary epithelial cells associated with mature stellate and stromal cells (ICAM-1+, ASMA +, vitamin a + +, collagen I +) results in cell aggregates that remain on the surface of the organ and are not effectively engrafted. In some embodiments, implantation of mature epithelial cells requires that they be paired with immature mesenchymal cells that produce MMPs necessary for implantation and migration.

According to one embodiment disclosed herein, floating aggregates of organoids, stem/progenitor cells (such as BTSCs and ELSMCs) are demonstrated to be the most successful cell presentation for patch implantation success. Disclosed herein are the following: BTSCs and ELSMCs can be self-selected to be organoids by: mature mesenchymal cells were removed by standard panning procedures against cells adherent to conventional culture dishes under serum-free conditions, and the remaining cells (those that were not adherent) were then cultured in low-adherence culture dishes and in serum-free defined medium. Organoids self-assemble under these conditions.

In another aspect, the present disclosure provides a method of forming an organoid by culturing a first type of cell (epithelial) with a second type of cell (mesenchymal), wherein the second type of cell is a lineage-appropriate partner of the first type of cell, removing mature cells adherent to a conventional culture dish by a panning procedure, and recovering the self-assembled organoid from a culture suspension on a low-adherent culture dish. The first type of cell may be an epithelial stem cell, a transient proliferative cell, a committed epithelial progenitor cell. The second type of cell may be a stem cell of a mesenchymal cell, a transient proliferative cell or a committed mesenchymal progenitor cell.

In some embodiments, for the patch implantation strategy to be successful, the donor cells and/or supporting mesenchymal cells must express multiple matrix metalloproteinases (hereinafter MMPs), particularly secreted forms of MMPs. Without being bound by theory, it is believed that the various isoforms of MMPs allow for the lysis of the bursa around the organ or tissue, followed by rapid migration of the donor cells to the host tissue. The disclosure herein provides the expression that early epithelial stem cells and/or ELSMCs express high levels of membrane-associated and/or secreted MMPs, whereas mature cells (e.g., hepatocytes) express low levels of secreted MMPs, even though they also express plasma membrane-associated MMPs. Implantation of such adult cells (e.g., hepatocytes, biliary epithelium, islets, intestinal epithelium, etc.) requires the following: the mesenchymal partner is the cellular source of MMPs (especially secreted forms of MMPs) if implantation is to take place. An alternative is to provide the relevant isoforms of MMPs in the biomaterial of the implant, i.e. their purified forms.

According to the present disclosure, the number of cells that can be implanted using a patch implant is very large (>10⁸) And depends on the size of the implant, the number and size of the organoids (or the number of cells-if not part of an organoid) (whether the donor cells are stem cells or mature cells), and the expression of secreted and membrane-associated MMPs (from epithelial and/or from mesenchymal cells). These findings are quite different from the limited number of cells that can be used for vascular delivery or injection implantation (e.g., 10)⁵-10⁶)。

Disclosed herein are the following: preparing the implant includes mixing the cells with a suitable biomaterial that can be rendered insoluble and keep the cells localized at the target site. In another aspect, the cells of the isolated internal organ may be solidified ex vivo within the biological material prior to introducing the cells into the host, or in the alternative, injected with a fluid substance and allowed to solidify in vivo. In another aspect, the biomaterial that can form a hydrogel or parallel insoluble complex can include hyaluronic acid or other non-sulfated or minimally sulfated glycosaminoglycans, thiol-modified sodium hyaluronate, or plant-derived materials (e.g., alginate). The trigger for curing may be any factor that initiates crosslinking of the matrix components or gelling of those components where gelling may occur. The crosslinking agent may comprise polyethylene glycol diacrylate or a disulfide bond-containing derivative thereof. Preferably, the insoluble complex of cells and biological material has a viscoelasticity in the range of about 0.1 to 200Pa, preferably about 0.1 to about 1Pa, about 1 to about 10Pa, about 10-100Pa, or about 100 to about 200.

Preferably, the cells are introduced at or near diseased or dysfunctional tissue and may be introduced via injection or surgical delivery. Without being bound by theory, it is hypothesized herein that a thicker HA hydrogel (e.g., >500Pa) triggers cell differentiation and decreases engraftment, due in part to decreased expression of MMPs with maturation and a parallel decrease in migratory capacity.

Substrate

There are a number of options for a biocompatible, biodegradable substrate that is neutral to the mature state of the donor cells. They include silk forms of silk moth (such as Seri)^RSurgical Silk stents or profiled Seri-Silk (Sofregen, New York, NY)), other derivatives of Silk of bombyx mori, amnion derivatives, omentum, placenta, and synthetic fabrics or materials (such as in the form of polyglycolic acid-poly-L-lactic acid copolymer (PGA/PLLA)). The key to the effectiveness of the substrate is that it has minimal impact on the differentiation of the donor cells. Thus, many basal forms used clinically cannot be used for patch implantation because they are composed of components that induce donor cell differentiation (e.g., mature-type forms of extracellular matrix).

The substrate must have sufficient tensile strength to allow the implant to be attached to the target site by sutures or by surgical glue. It should consist of biocompatible, biodegradable materials that can degrade within months, but the degradation products do not change the maturation state of the donor cells. Thus, the product should have minimal impact on pH or other aspects of the environment. The substrate must also be able to conform to the surface of the target site; the flexible substrate will facilitate the use of the implant at a site of significant bending. Seri-Silk is a non-limiting example of a suitable material for the substrate. Amnion-derived substitutes for materials suitable for use as substrates are also contemplated, such as, but not limited to, amnion-derived materials produced by Osiris Therapeutics, Inc (Columbia, MD).

The substrate may be derived from a porous scaffold, such as Seri-silk, or a non-porous membrane, such as an amniotic membrane or a placental membrane or omentum, or may be a porous or non-porous synthetic fabric, or a combination thereof. If the substrate is porous, it is sealed using a biomaterial infusion/impregnation to inhibit migration of the cell population in the direction of the substrate (i.e., away from the target site, or through the substrate). As defined above, the key features of the base material are biocompatibility, biodegradability neutral, and sufficient tensile strength as described above. In addition, the material may optionally be bioresorbable.

The substrate may be further optimized according to the application. For example, in some embodiments, if the patch implant includes a substrate designed to survive under the influence of the air drying effect, it may be applied to the skin and subcutaneous dermal tissue.

The hydrogel matrix disclosed above may also be used in other parts of a patch implant. For example, if the biocompatible, biodegradable substrate is porous, a hydrogel may be used to inhibit migration of the cell population in the direction of the substrate. Such hydrogels require higher viscoelasticity than hydrogels, e.g., between 1.5 and 15 times higher, e.g., 2 times higher. Non-limiting examples of suitable viscoelastics include, but are not limited to, viscoelastic properties in the range of from about 250 to about 600Pa, such as at least about 250Pa, at least about 300Pa, at least about 350Pa, at least about 400Pa, at least about 450Pa, at least about 500Pa, at least about 550Pa, at most about 600Pa, at most about 550Pa, at most about 500Pa, at most about 450Pa, at most about 400Pa, at most about 350Pa, at most about 200Pa, and/or any single value therebetween, such as, but not limited to, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, and about 600 Pa. Other non-limiting examples of suitable viscoelastics include, but are not limited to, viscoelastics in the range of from about 600 to about 800Pa, such as at least about 600Pa, at least about 650Pa, at least about 700Pa, at least about 750Pa, at most about 800Pa, at most about 750Pa, at most about 700Pa, at most about 650Pa, at most about 600Pa, and/or any single value therebetween, such as, but not limited to, viscoelastics of about 600Pa, about 650Pa, about 700Pa, about 750Pa, and about 800 Pa. Additional non-limiting examples include a range of about 250Pa to about 800 Pa.

Still further, the hydrogels disclosed herein may be used as a coating to prevent adhesion on the serosal surface of the substrate, which is opposite the substrate side of the adjacent cells. Such hydrogels may have viscoelasticity between that suitable for use with cell-containing hydrogels and suitable sealing substrates. Non-limiting examples of suitable viscoelastics include, but are not limited to, viscoelastics in the range of from about 250 to about 400Pa or about 500Pa, e.g., at least about 250Pa, at least about 300Pa, at least about 350Pa, at least about 400Pa, at least about 450Pa, at most about 500Pa, at most about 450Pa, at most about 400Pa, at most about 350Pa, at most about 200Pa, and/or any single value therebetween, such as, but not limited to, viscoelastics of about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, and about 500 Pa.

Implant, general description

In general, patch implants can be designed using the aforementioned methods and components for transplantation of donor (allogeneic or autologous) cells to a solid organ or tissue, as well as conditions to maintain and maintain the donor cells in an early mature lineage stage. In particular, patch implants for the transplantation of donor cells (allogeneic or autologous) into a solid organ or tissue, as well as conditions that maintain and maintain some or all of the donor cells in the early mature lineage stage, are contemplated. In some embodiments, the donor cell is a mixture of epithelial and mesenchymal cells. In some embodiments, both donor cell populations are stem/progenitor cells. In some embodiments, the epithelial cells are mature cells (e.g., hepatocytes, islets, etc.) and the mesenchymal cells are stem/progenitor cells. In some embodiments, the conditions of the implant biomaterial, such as the culture medium and the matrix components, maintain both donor cell populations, or at least the mesenchymal cell population, as stem/progenitor cells. In some embodiments, the culture medium comprises a basal medium and a soluble signal. In further embodiments, the basal medium and soluble signal support sternness maintenance in both donor populations or at least in the mesenchymal cell population. In some embodiments, the matrix (optionally including extracellular matrix components) and its consistency level support the maintenance of the sternness of the donor population or at least the mesenchymal cell population. In some embodiments, the matrix comprises hyaluronic acid, optionally prepared as a soft hydrogel having a viscoelasticity of about 50Pa to about 150 Pa. In some embodiments, the patch implant includes a substrate having sufficient mechanical strength to tether the implant to the target site, and is composed of a biocompatible, biodegradable material that does not significantly alter the mature lineage stage of the donor cells. Optionally, without further modification, the substrate itself should be sufficient to protect the layer containing donor cells that do not significantly affect the mature lineage stage of the donor cells. In some embodiments, the substrate is a mesh or scaffold and is further impregnated with a biomaterial (such as hyaluronic acid) that has sufficiently high viscoelastic properties to allow sufficient maturation of any cells that migrate thereto, thereby eliminating migration of donor cells in directions other than toward the target site. In some embodiments, the viscoelastic properties are about 500Pa or greater. In some embodiments, the serological surface of the implant is coated with a biomaterial to minimize adhesion from adjacent tissues or organs. In some embodiments, the biomaterials have a viscoelasticity of about 200Pa to about 300 Pa.

The proposed substrate is envisioned to be elastic enough to withstand mechanical forces, capable of tethering to a target organ or tissue, and flexible enough to tether into a position with bending. Any biomaterial (other than hydrogel) may also be employed, provided that the biomaterial is capable of retaining and maintaining a cell population and has viscoelastic properties sufficient to allow migration of the cell population within or away from the patch implant.

In another embodiment, a patch implant may be used to hold and maintain a cell population and include: (a) a population of cells (optionally a population of cells of a single type) supported in a culture medium in a hydrogel or other biological material having sufficient viscoelastic properties to allow migration of cells within or away from the patch implant; and (b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of a cell population in the direction of the substrate (or to provide a barrier to migration of the cell population).

It is important to note that MMPs can be membrane-associated and/or secreted MMPs; they may be provided by MMP producing cells, derived from these cells, or they may be added to the composition of interest (e.g., a purified or recombinantly produced composition).

In another embodiment, a covering or coating for a patch implant or tissue is provided that includes a hydrogel or other biomaterial having sufficient viscoelasticity and elasticity to withstand the mechanical forces applied against the covering or coating (from or by other tissues and organs). By using a covering or coating, a method for inhibiting or preventing the formation of adhesions (which may involve or result from mechanical forces or contact from other organs and tissues) is provided, which comprises covering or coating a surface with hydrogel or other comparable biological material.

In yet another embodiment, a method of implanting cells into a target tissue is provided, the method comprising contacting the target tissue with a patch implant comprising: (a) a population of cells, including at least one population having an early lineage stage, including a single type or multiple types of cells supported in a hydrogel or other biological material having rheological properties (e.g., viscoelasticity) sufficient to allow the cells of the population to migrate within or away from the patch implant; and (b) a substrate comprising a biocompatible, biodegradable material having rheological properties (e.g., viscoelasticity) sufficient to inhibit migration of cells of a population in the direction of the substrate (or to provide a barrier to migration of cells of a population), the patch implant being configured to retain and maintain the population of cells while inhibiting differentiation or further maturation of the at least one population having an early lineage stage to a late lineage stage. In another embodiment, there is provided a method of: wherein a population with an early lineage stage is capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs). In another embodiment, the cell does not have this capability, but MMPs from other sources (e.g., recombinant) are present or included.

Implants with cellular sources of MMPs

Aspects of the present disclosure relate to a patch implant for retaining and maintaining a mixed population of cells, the patch implant comprising (a) a mixed population of two or more cell types, at least one type of which is at an early lineage stage capable of expressing secreted and/or membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), the mixed population being supported in a culture medium present in a hydrogel matrix having sufficient viscoelastic properties to allow migration of the mixed population (optionally, within or away from the hydrogel and/or within or away from the patch implant); (b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in the direction of the substrate; and optionally (c) a hydrogel overlying a serosal (i.e., external) surface of the substrate, the serosal surface opposite the surface contacting the mixed population and, in embodiments where the patch implant is tethered to a target site, opposite the side contacting the target site (e.g., (organ or tissue) — in some embodiments, this layer prevents or inhibits adhesions by, or prevents or inhibits adhesions with, other tissues or organs.

In some embodiments, the implant may contain only one cell type, such as Embryonic Stem (ES) cells or Induced Pluripotent Stem (iPS) cells. Success can be achieved as long as these cells are a cellular source of MMPs, or other sources, such as purified (e.g., recombinant) forms of MMPs, are added to the implant.

In some embodiments, the substrate is porous or non-porous. In some embodiments, the substrate comprises a porous and/or non-porous mesh, scaffold, or membrane. In some embodiments, the substrate comprises a filament; a synthetic fabric; or a natural material (such as amnion, placenta or omentum or derivatives thereof); or a combination thereof. In some embodiments, the substrate comprises a porous mesh infused with a hydrogel or other biological material that is used to convert to a barrier. In further embodiments, such infusion prevents migration of cells away from the target organ or tissue. In some embodiments, the substrate comprises a solid material.

In some embodiments, one or more of the hydrogels comprises hyaluronic acid.

In some embodiments, the culture medium comprises a depot column medium or another medium that supports stem cells and is capable of maintaining a dry quality.

In some embodiments, the mixed population comprises mesenchymal cells and epithelial cells. In some embodiments, the epithelial cells can be ectodermal, endodermal, or mesodermal cells. In some embodiments, the mesenchymal cells comprise Early Lineage Stage Mesenchymal Cells (ELSMCs). In some embodiments, the ELSMC includes one or more of a hemangioblast, a precursor of endothelium, a precursor of stellate cells, and a Mesenchymal Stem Cell (MSC). In some embodiments, the epithelial cells comprise epithelial stem cells. In some embodiments, the epithelial cells comprise biliary stem cells (BTSCs). In some embodiments, the epithelial cells comprise committed and/or mature epithelial cells. In some embodiments, the committed and/or mature epithelial cells comprise mature parenchymal cells. In some embodiments, the mature parenchymal cells include one or more of hepatocytes, cholangiocytes, and islet cells. In some embodiments, the mesenchymal cells and epithelial cells both comprise stem cells.

In a certain embodiment, the mixed population comprises autologous cells and/or allogeneic cells.

In some embodiments, one or more cell types are genetically modified.

"layered" implant

In some embodiments, the patch implant is considered a multi-layered implant. For example, provided herein are patch implants comprising, consisting of, or consisting essentially of a plurality of layers, the plurality of layers comprising at least: (a) a soft hydrogel first layer comprising donor cells, optionally epithelial and/or mesenchymal cells; (b) a thickened hydrogel second layer; and (c) a third layer comprising a biocompatible, biodegradable substrate. In some embodiments, particularly where the third layer is porous, the second layer is incorporated into, impregnated into, and/or infused into the third layer. In some embodiments, the patch implant further comprises a fourth layer of hydrogel. In some embodiments of the patch implant, the fourth layer is coated or painted onto the serosal surface of the implant. In some embodiments of the patch implant, the first layer is adapted to directly contact the target tissue or organ.

As used herein, "soft" refers to a hydrogel layer that exhibits a low level of internal pressure as quantitatively determined by the pascal (Pa) assay. One pascal is defined as one newton per square meter. In some embodiments, the softer layer has a viscosity of from about 10Pa to about 300Pa, from about 50Pa to about 250Pa, from about 100Pa to about 250Pa, from about 50Pa to about 200Pa, from about 150Pa to about 200Pa, or from about 100Pa to about 200 Pa. In a particular embodiment, the soft hydrogel layer has a viscosity of less than or about equal to 200 Pa.

As used herein, "thick" refers to a hydrogel layer that exhibits a high level of internal pressure as quantitatively determined by the pascal (Pa) assay. In some embodiments, the thick stock layer has a viscosity of about 300Pa to about 3000Pa, about 300Pa to about 1000Pa, about 400Pa to about 750Pa, about 400Pa to about 550Pa, about 450Pa to about 600Pa, or about 500Pa to about 600 Pa. In a particular embodiment, the thick hydrogel layer has a viscosity greater than or about equal to 500 Pa.

Preferably, for the first layer of the layered implant, the insoluble complex of cells and biomaterial has a viscosity or viscoelasticity in the range of about 0.1 to 200Pa, preferably about 0.1 to about 1Pa, about 1 to about 10Pa, about 10 to 100Pa, or about 100 to about 200, or about 50 to about 250Pa, or about 200 Pa. Preferably, for the first layer of the layered implant, the insoluble complex of cells and biological material has a viscoelasticity in the range of about 0.1 to 200Pa, preferably about 0.1 to about 1Pa, about 1 to about 10Pa, about 10 to 100Pa, or about 100 to about 200.

In some embodiments, the one or more cells in the mixture are a source of secreted and/or membrane-associated MMPs. In some embodiments, such as but not limited to those involving stem/progenitor cell populations that naturally secrete MMPs, the variable that silences MMP expression-optionally secreted MMP expression-is controlled in the patch implant. Non-limiting examples of such variables include variables that lead to stem/progenitor cell maturation such as, but not limited to, serum supplements to culture media or implant biomaterials, hormones or other soluble signals that affect differentiation of epithelial and/or mesenchymal cells, oxygen levels (since anaerobic conditions keep cells in an immature state, while higher oxygen levels promote differentiation), and rigidity of implant materials (since mechanical forces (such as shear forces) and compression can drive differentiation).

In some embodiments of the patch implant, the viscosity of the first layer is about 50 to about 250 Pa. In some embodiments of the patch implant, the viscosity of the first layer is about 200 Pa. In some embodiments of the patch implant, the second layer has a viscosity of about 250Pa to about 600 Pa. In some embodiments of the patch implant, the viscosity of the second layer is about 500 Pa. In some embodiments of the patch implant, the viscosity of the fourth layer is from about 250 to about 500 Pa. In some embodiments of the patch implant, the viscosity of the fourth layer is about 400 Pa. In some embodiments of the patch implant, the viscosity of the second layer is greater than the viscosity of the 1 st layer. In some embodiments of the patch implant, the viscosity of the second layer is about 1.5 to about 15 times greater than the viscosity of the first layer. In some embodiments of the patch implant, the second layer has a viscosity of about 2 times greater than the first layer.

In one embodiment, the patch implant comprises, consists of, or consists essentially of a plurality of layers, starting from where it contacts the target site, and consisting of: donor cells embedded in a soft (<200Pa) hydrogel prepared in serum-free defined medium (these cells will engraft and migrate into the tissue); a second layer of hydrogel prepared in the same medium and triggered to have a higher consistency (e.g., -500 Pa or higher) to provide a barrier to migration of donor cells in any direction other than towards the target tissue; a third layer of biocompatible, biodegradable, bioresorbable substrate that is neutral in its effect on the maturation state of the donor cells and that can be used intra-operatively or by other means to tether the implant to the target site; and a final layer of hydrogel having a consistency intermediate between that of the soft hydrogel and the very thick hydrogel and being sufficiently fluid to coat or coat a surface to minimize adhesion near tissue.

In some embodiments of the patch implant, the first layer and the second layer each comprise one or more hyaluronic acids. In some embodiments of the patch implant, the fourth layer comprises one or more hyaluronic acids.

In some embodiments of the patch implant, the epithelial cells and mesenchymal cells form one or more aggregates. In some embodiments of the patch implant, the one or more aggregates are organoids. In some embodiments of the patch implant, the epithelial cells comprise epithelial stem cells. In some embodiments of the patch implant, the epithelial cells comprise biliary epithelial cells. In some embodiments of the patch implant, the epithelial cells comprise committed and/or mature epithelial cells. In some embodiments of the patch implant, the committed and/or mature epithelial cells comprise mature parenchymal cells. In some embodiments of the patch implant, the mature parenchymal cells comprise one or more of hepatocytes, cholangiocytes, and islet cells.

In some embodiments of the patch implant, the mesenchymal cells are supportive mesenchymal cells. In some embodiments of the patch implant, the mesenchymal cells comprise early lineage mesenchymal cells (ELSMCs). In some embodiments of the patch implant, the ELSMC includes one or more selected from the group consisting of angioblasts, precursors of endothelium, precursors of stellate cells, and Mesenchymal Stem Cells (MSCs).

In some embodiments of the patch implant, the epithelial cells and the mesenchymal cells are not lineage-collocated with each other. In some embodiments of the patch implant, the epithelial cells are mature cells. In some embodiments of the patch implant, the mesenchymal cells are ELSMCs.

In some embodiments of the patch implant, at least one of the epithelial cells and the mesenchymal cells are derived from a donor. In some embodiments, the donor is a subject in need of tissue transplantation. In some embodiments, the donor is a source of healthy cells for tissue transplantation. In some embodiments of the patch implant, at least one of the epithelial cells and the mesenchymal cells are autologous to the intended recipient of the patch implant. In some embodiments, all cells (i.e., epithelial and mesenchymal cells) are autologous to the intended recipient of the implant. In some embodiments, the donor of the cells may be a donor other than the recipient (allogeneic), or may also be a subject with an internal organ that is diseased or dysfunctional (autologous), optionally wherein the donor is obtained from a portion of the internal organ that is not diseased or dysfunctional and/or has been genetically modified to restore function to the cells. For establishing a model system for studying disease, the donor cells may be cells that have the disease and are transplanted onto/into normal tissue in the experimental host.

In some embodiments of the patch implant, at least one of the epithelial cells or mesenchymal cells are modified. In some embodiments, all cells are modified. In some embodiments, the modification is a genetic modification. In some embodiments, the one or more cells are modified to express a therapeutic nucleic acid or polypeptide. In some embodiments, the one or more cells are modified to express a wild-type allele of a nucleic acid or polypeptide.

In some embodiments of the patch implant, the biocompatible, biodegradable substrate is bioresorbable. In some embodiments of the patch implant, the biocompatible, biodegradable substrate comprises a porous material. In some embodiments of the patch implant, the biocompatible, biodegradable substrate comprises a scaffold or a membrane. In some embodiments of the patch implant, the scaffold or membrane comprises silk, amniotic membrane, synthetic fabric, or a combination thereof. In some embodiments, the biocompatible, biodegradable substrate does not include any factors that induce or prevent cell differentiation. In some embodiments of the patch implant, the biocompatible, biodegradable substrate does not include one or more components derived from the mature extracellular matrix. In some embodiments of the patch implant, the component derived from the mature extracellular matrix is type I collagen.

In some embodiments of the patch implant, the patch implant further comprises one or more Matrix Metalloproteinases (MMPs). In some embodiments of the patch implant, the MMPs are membrane-associated MMPs. In some embodiments of the patch implant, the membrane-associated MMPs are provided by one or more of epithelial cells or mesenchymal cells. In some embodiments of the patch implant, the MMP is a secreted MMP. Secreted MMPs may optionally be naturally produced by one or more of epithelial or mesenchymal cells, or optionally be produced as a result of transformation of one or more of epithelial or mesenchymal cells with a recombinant expression vector for MMP production.

In some aspects, provided herein are patch implants comprising, consisting of, or consisting essentially of a plurality of layers, the plurality of layers comprising at least: a soft hydrogel first layer comprising biliary stem cells; a thickened hydrogel second layer; and a third layer comprising a biocompatible, biodegradable substrate.

In one embodiment, the patch implant is composed of layers of material and cells that together form a "bandage-like implant" that can be surgically or otherwise tethered to a target site. The first layer next to the target site comprises a soft hydrogel (less than 200Pa) seeded with a mixture of epithelial cells and supporting mesenchymal cells suspended in defined serum-free nutrient-rich medium designed for the expansion and/or survival of the cells; the second layer contains a hydrogel that is prepared in the same medium but gels to a thicker level (i.e., higher pascal level) and forms a barrier that blocks migration of cells in directions other than the target site; the third level comprises a biocompatible, biodegradable substrate that does not affect or minimally affects the level of differentiation of the donor cells, but acts as a mechanical support structure for the patch; a fourth layer consisting of a coatable hydrogel (such as hyaluronic acid in addition) having a consistency level intermediate between that of the soft hydrogel and the thick hydrogel and serving to minimize adhesion from cells from adjacent tissues to the implant. Hydrogels must be composed of biocompatible, biodegradable and "tunable" (meaning controllable with respect to consistency) materials. One successful material for hydrogels is thiol-modified hyaluronic acid, which can be triggered to form hydrogels when exposed to oxygen and/or poly (ethylene glycol) diacrylate (PEGDA), and can be easily "tuned" by the precise ratio of hyaluronic acid and PEGDA concentrations (and/or oxygen levels).

In another embodiment, the patch implant includes multiple layers. The first layer next to the target site is a soft hydrogel, which is a minimally sulfated or non-sulfated GAG or other non-sulfated or neutral biomaterial that can be gelled or solidified and in the middle can place the donor cell. The second layer of hydrogel or biomaterial is thicker and incorporates a substrate, i.e., a biocompatible, biodegradable, bioresorbable substrate that allows the patch to be handled for surgery or other purposes and acts as a barrier to forcing cells to migrate toward the target tissue, in/on or within. The serosal side of the substrate is surgically coated with a biomaterial, such as hyaluronic acid (or other minimally sulfated or non-sulfated GAGs or other materials that may gel or solidify), and wherein the pascal level is at least twice that present in the soft biomaterial layer; this serves the purpose of minimizing adhesions from adjacent tissue. The patch implant is tethered to the target organ or tissue and the cells are able to migrate to and fully incorporate into the tissue or organ.

In a particular embodiment, the patch implant comprises a first layer of soft biomaterial (<200Pa), such as soft hyaluronic acid hydrogel, in between which are placed donor cells to be transplanted in serum-free defined medium, tailored for the lineage stage of the cells. This layer is placed on a thicker layer (e.g., a thicker hydrogel) that acts as a barrier to force donor cells to be directed during migration to the target tissue. The thicker layer is prepared in advance on a substrate (i.e., a biocompatible, biodegradable substrate that allows the patch to be processed for surgery or other procedures to secure the patch to the target site). The last layer is a biomaterial with a consistency between that against the donor cells on the target tissue side and that against the barrier. This layer is added to the serosal side of the implant at the time of surgery and serves to minimize adhesions from adjacent tissue. The biocompatible, biodegradable substrate may be Seri-silk or derivatives thereof.

Methods of use and delivery of patch implants

Aspects of the present disclosure relate to compositions and methods for implanting cells into an organ. The task of transplanting cells from a solid organ to an internal organ typically uses direct injection or delivery of cells via the vascular pathway. Lanzoni, G, et al, Stem Cells 31,2047-2060 (2013). These transplantation methods produce small numbers of cells that are transplanted to the target site and carry the risk of potentially life-threatening emboli. If the cells are delivered by "injection implantation", in which the cells are suspended in or coated with hyaluronic acid, and then co-injected with an initiator (PEGDA) that causes the hyaluronic acid to gel in situ, the cells are then injected in situGrafting was improved as described in Turner R et al Hepatology 57,775- & 784 (2013). The injection implantation method provides a strategy for localizing cells to a specific site, although in small numbers, typically 10⁵-10⁷、10⁶-10⁷Or 10⁵-10⁶Individual cells/injection site. This strategy eliminates or minimizes ectopic cell distribution and optimizes cell integration in the site. However, if mature functional cells are used, they may be highly immunogenic, which requires long-term immunosuppression. In addition, the number of cells that can be injected may not be sufficient to achieve the necessary clinical results.

The "patch implantation" strategy described herein overcomes these obstacles and problems. In some embodiments, a "bandage-like" implant is surgically or otherwise tethered to the surface of an organ or tissue; the conditions of the implant allow cells to be fully implanted in the site, migrate throughout the organ/tissue, and then mature into the relevant adult cell type. The large number of cells is formed or determined by the size of the patch, the number or mixture of cells within the implant, and the source of the various forms of MMP (ideally a cellular source of MMPs) ((ii))>10⁸Individual cells). Furthermore, in some embodiments, the use of organoids aids in the ability to store donor cells, and the manner in which organoids are cryopreserved under defined serum-free conditions is more conducive to such ability.

The patch implant compositions provided herein relate to the direct implantation of cells into a tissue or solid organ. The method is safe, avoids embolism and ectopic cell distribution, and optimizes cell number implantation and distribution in and throughout the tissue.

Accordingly, provided herein is a method of implanting cells into a target tissue, the method comprising, consisting of, or consisting essentially of contacting the target tissue with the patch implant disclosed above.

In some embodiments of the method, the target tissue is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, gastrointestinal tract, lung, prostate, breast, brain, bladder, spinal cord, skin and subcutaneous dermal tissue, uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and skeletal tissue. In some embodiments of the method, the target tissue is liver tissue. In some embodiments of the method, the target tissue is pancreatic tissue. In some embodiments of the method, the target tissue is biliary tissue. In some embodiments of the method, the target tissue is a gastrointestinal tract tissue. In some embodiments, the tissue is diseased, damaged, or has a disorder. In some embodiments of the method, the target tissue is kidney tissue.

In some embodiments of the method, the target tissue is an organ. In some embodiments of the method, the organ is an organ of the musculoskeletal system, digestive system, respiratory system, urinary system, female reproductive system, male reproductive system, endocrine system, circulatory system, lymphatic system, nervous system, or integumentary system. In some embodiments of the method, the organ is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, gastrointestinal tract, lung, prostate, breast, brain, bladder, spinal cord, skin and subcutaneous dermal tissue, uterus, kidney, muscle, blood vessels, heart, cartilage, tendons, and bone. In some embodiments, the organ is diseased, damaged, or has a disorder.

In some embodiments of the method, the liver disease or disorder is liver fibrosis, cirrhosis, hemochromatosis, liver cancer, biliary atresia, nonalcoholic fatty liver disease, hepatitis, viral hepatitis, autoimmune hepatitis, fascioliasis, alcoholic liver disease, α 1-antitrypsin deficiency, glycogen storage disease type II, transthyretin-associated hereditary amyloidosis, Gilbert's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, bade-Chiari syndrome, liver trauma, or Wilson's disease.

In other aspects, provided herein are methods of treating a subject having a pancreatic disease or disorder, comprising, consisting of, or consisting essentially of contacting a pancreas of the subject with the patch implant disclosed above. In some embodiments of the method, the pancreatic disease or disorder is diabetes, pancreatic exocrine insufficiency, pancreatitis, pancreatic cancer, Oddi (Oddi) sphincter dysfunction, cystic fibrosis, pancreatic division, cricoid pancreas, pancreatic trauma, or pancreatic ductal hemorrhage.

In other aspects, provided herein are methods of treating a subject having a disease or disorder of the gastrointestinal tract, comprising, consisting of, or consisting essentially of contacting one or more intestines of the subject with a patch implant disclosed above. In some embodiments, the gastrointestinal disease or disorder is gastroenteritis, gastrointestinal cancer, ileitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, peptic ulcer disease, celiac disease, fibrosis, vascular dysplasia, hirschsprong's disease, pseudomembranous colitis, or gastrointestinal trauma.

In some aspects, provided herein are methods of treating a subject having a kidney disease or disorder, the method comprising, consisting of, or consisting essentially of contacting one or more kidneys of the subject with a patch implant disclosed above. In some embodiments of the method, the kidney disease or disorder is nephritis, nephropathy, nephritic syndrome, nephrotic syndrome, chronic nephropathy, acute kidney injury, kidney trauma, cystic nephropathy, polycystic nephropathy, glomerulonephritis, IgA nephropathy, lupus nephritis, kidney cancer, Alport (Alport) syndrome, amyloidosis, Goodpasture's syndrome, or Wegener's granulomatosis.

In some embodiments of the method of therapy, at least one of the epithelial cells and the mesenchymal cells are derived from a donor. In some embodiments, the donor is a subject in need of tissue transplantation. In some embodiments, the donor is a source of healthy cells for tissue transplantation. In some embodiments, at least one of the epithelial cells and the mesenchymal cells is autologous to the intended recipient of the patch implant. In some embodiments, all cells (i.e., epithelial and mesenchymal cells) are autologous to the intended recipient of the implant. In some embodiments, the donor of the cells may be a donor other than the recipient (allogeneic), or may also be a subject with an internal organ that is diseased or dysfunctional (autologous), optionally wherein the donor is obtained from a portion of the internal organ that is not diseased or dysfunctional and/or has been genetically modified to restore function to the cells.

In some embodiments, the patch implant used in the method disclosed above is a patch implant comprising a plurality of layers, the plurality of layers comprising at least: a hydrogel first layer comprising epithelial cells and mesenchymal cells; a second layer of hydrogel; a third layer comprising a biocompatible, biodegradable substrate; and optionally a fourth layer of hydrogel. In some embodiments, the method further comprises allowing cells contained in the patch implant to incorporate into the tissue. In some embodiments of the method, the hydrogel first layer is soft. In some embodiments of the method, the hydrogel second layer is a thick. In some embodiments of the method, the mesenchymal cells are supportive mesenchymal cells.

In another aspect, the present disclosure provides a method for implanting cells into an organ, the method comprising the use of a patch implant (i.e., a bandage-like composite having multiple layers of materials and cells) that together may be surgically or otherwise tethered to a target site. The first layer next to the target site comprises a soft hydrogel (less than 200Pa) seeded with a mixture of epithelial cells and supporting mesenchymal cells suspended in defined serum-free nutrient-rich medium designed for the expansion and/or survival of the cells; the second layer contains a hydrogel that is prepared in the same medium but gels to a thicker level (i.e., higher pascal level) and forms a barrier that blocks migration of cells in directions other than the target site; the third level comprises a biocompatible, biodegradable substrate that does not affect or minimally affects the level of differentiation of the donor cells, and is therefore "neutral"; a fourth layer consisting of a coatable hydrogel (such as hyaluronic acid in addition) having a consistency level intermediate between that of the soft hydrogel and the thick hydrogel and serving to minimize adhesion from cells from adjacent tissues to the implant. Hydrogels must be composed of biocompatible, biodegradable and "tunable" (meaning controllable with respect to consistency) materials. One successful material for hydrogels is thiol-modified hyaluronic acid, which can be triggered to form hydrogels when exposed to oxygen and/or poly (ethylene glycol) diacrylate (PEGDA), and can be easily "tuned" by the precise ratio of hyaluronic acid and PEGDA concentrations (and/or oxygen levels). The cells produce a variety of Matrix Metalloproteinases (MMPs) under the conditions of the biomaterial of the implant that facilitate implantation, migration and integration of the donor cells into the recipient's tissue. The microenvironment of the recipient tissue determines the adult fate of the transplanted cells.

In another aspect, the present disclosure provides a method for implanting cells into an organ, the method comprising contacting a patch implant, the patch implant comprising a plurality of layers, the plurality of layers comprising at least a first layer comprising a biocompatible, biodegradable substrate, a second layer comprising one or more hyaluronic acids comprising a mixture of epithelial cells and supporting mesenchymal cells, and a third layer comprising one or more hyaluronic acids, wherein the layer with cells embedded in the middle is soft (less than 200 Pa); the layers associated with the substrate are thicker (-500 Pa or greater); the pascal level of the third layer is intermediate and helps to minimize adhesion near the tissue or organ. In yet another aspect, the cells can be implanted in an organ selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, intestine, lung, prostate, breast, brain, spinal cord, ganglia, skin and subcutaneous dermal tissue, uterus, bone, thymus, intestine, uterus, bone, kidney, muscle, blood vessels, or heart.

In yet another aspect, the cells may be implanted in an organ selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, intestine, lung, prostate, breast, brain, spinal cord, ganglia, skin and subcutaneous dermal tissue, uterus, bone, tendons, cartilage, kidney, muscle, blood vessels, or heart.

Non-limiting examples of patch implants suitable for use in the methods disclosed herein are patch implants comprising: (a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing secreted and/or membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), the mixed population being supported in a culture medium present in a hydrogel matrix having sufficient viscoelastic properties to allow migration of the mixed population (optionally, within or away from the hydrogel and/or within or away from the patch implant); (b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in the direction of the substrate or to provide a barrier to said migration; and optionally (c) a hydrogel overlying a serosal (i.e., external) surface of the substrate, the serosal surface opposite the surface contacting the mixed population and, in embodiments where the patch implant is tethered to a target site, opposite the side contacting the target site (e.g., (organ or tissue) — in some embodiments, this layer prevents or inhibits adhesions by, or prevents or inhibits adhesions with, other tissues or organs.

In some embodiments, the substrate is porous or non-porous. In some embodiments, the substrate comprises a porous mesh, scaffold, or membrane. In some embodiments, the substrate comprises a filament; a synthetic fabric; or natural materials (such as amnion, placenta or omentum); or a combination thereof. In some embodiments, the substrate comprises a porous mesh infused with a hydrogel. In further embodiments, such infusion prevents migration of cells away from the target organ or tissue. In some embodiments, the substrate comprises a solid material.

In some embodiments, the patch implant further comprises a hydrogel superimposed on a serosal surface of the substrate, the serosal surface being opposite the surface contacting the single cell or mixed population of cells.

In some embodiments, one or more of the hydrogels comprises hyaluronic acid.

In some embodiments, the culture medium comprises a depot column medium or another medium that supports stem cells and is capable of maintaining a dry quality.

In some embodiments, the mixed population comprises mesenchymal cells and epithelial cells. In some embodiments, the epithelial cells can be ectodermal, endodermal, or mesodermal cells. In some embodiments, the mesenchymal cells comprise Early Lineage Stage Mesenchymal Cells (ELSMCs). In some embodiments, the ELSMC includes one or more of a hemangioblast, a precursor of endothelium, a precursor of stellate cells, and a Mesenchymal Stem Cell (MSC). In some embodiments, the epithelial cells comprise epithelial stem/progenitor cells. In some embodiments, the epithelial cells comprise biliary stem cells (BTSCs). In some embodiments, the epithelial cells comprise committed and/or mature epithelial cells. In some embodiments, the committed and/or mature epithelial cells comprise mature parenchymal cells. In some embodiments, the mature parenchymal cells include one or more of hepatocytes, cholangiocytes, and islet cells. In some embodiments, the mesenchymal cells and epithelial cells both comprise stem cells.

In a certain embodiment, the mixed population comprises autologous cells and/or allogeneic cells.

In some embodiments, one or more cell types are genetically modified.

Examples

The following examples are non-limiting and illustrate procedures that may be used in various circumstances to effect the present disclosure. In addition, all references disclosed below are incorporated by reference in their entirety.

Example 1: pig model for patch implant verification

Animal(s) production

Animals used as hosts or as donors of cells were maintained in facilities of the NCSU (Raleigh, NC) veterinary college. Surgery, autopsy, and collection of all biological fluids and tissues are performed in these facilities. All procedures were approved by the NCSU IACUC committee. Pigs used as recipients were a mixture of six different breeds: six breed hybrids consisting of Yorkshires, Large Whits, Landraces (from sows), Durocs, Spots, and Pietrans (from boars). These highly heterogeneous genetic backgrounds are desirable because they correspond to heterogeneous genetic constructs of the human population. The host animals were all female, approximately six weeks of age, weighing-15 kg.

There are two classes of host animals, a) male pigs (approximately six weeks of age, weighing-15 kg) were used as donors for cell transplantation to female pigs, b) transgenic donor animals carrying a GFP transgene the GFP + donor animals were obtained by crossing transgenic H2B-GFP male pigs with wild type gilts by standard artificial insemination the model was developed via IRES-pH2B-eGFP to endogenous β -Actin (ACTB) locus CRISPR-Cas9 mediated Homology Directed Repair (HDR).

For each donor and recipient animal, the porcine leukocyte antigen class I (SLA-I) and class II (SLA-II) loci have been PCR amplified using primers designed to amplify known alleles in these regions according to a PCR sequence-specific primer strategy. The system consists of amplifying the gene loci of SLA-1, SLA-2 and SLA-3⁵³And 47 discriminatory SLA-I primer sets for amplifying DRB1, DQB1 and DQA loci. These primer sets have been developed to differentiate alleles by groups sharing similar sequence motifs, and have been readily and unambiguously shown to detect known SLA-I and SLA-II alleles. When used together, these primer sets are effective in providing a haplotype for each test animal, thereby providing an assay that readily confirms the matched or mismatched haplotypes in the donor and recipient animals.

Culture media and solutions

All media were sterile filtered (0.22 μm filter) and stored at 4 ℃ protected from light prior to use. Basal medium and Fetal Bovine Serum (FBS) were purchased from GIBCO/Invitrogen. All growth factors were purchased from R & D Systems. All other reagents were obtained from Sigma, except as already indicated.

The cell detergent was formulated with 599ml basal medium (e.g., RPMI 1640; Gibco # 11875-. It is used to wash tissue and cells during treatment.

A collagenase buffer consisting of 100ml of a cell detergent supplemented with collagenase (Sigma # C5138) (final concentration of 600U/ml (R145125 mg) for biliary (bile duct) tissue and 300U/ml (12.5mg) for organ parenchymal tissue (liver, pancreas)) was prepared.

Stock tower medium (defined serum-free medium designed for endodermal stem/progenitor cells) was used to prepare cell suspensions, organoids, and HA hydrogels. The medium was composed of copper-free, small amount of calcium (0.3mM), 1nM selenium, 0.1% bovine serum albumin (purified)No fatty acid; fraction V), 4.5mM nicotinamide, 0.1nM zinc sulfate heptahydrate, 5 μ g/ml transferrin/Fe, 5 μ g/ml insulin, 10 μ g/ml high density lipoprotein and a mixture of purified free fatty acids (provided in complex with fatty acid free, highly purified albumin) in any basal medium (here RPMI 1640). Its preparation is detailed in a summary of the methods⁵⁷. In addition, it is commercially available from Phoenix Songs Biologicals (Branford, CT).

Soluble long-chain form of HA (Sigma catalog #52747) is used for organoid culture stabilization and cryopreservation. Those HA (thiol-modified HA) used to prepare hydrogels were obtained from Glycosan biosciences, a subsidiary of Biotime. These thiol-modified HA components were prepared by proprietary bacterial fermentation processes using Bacillus subtilis as host in ISO 9001:2000 process (www.biopolymer.novozymes.com /). These components are sold under the trade name Novozymes

Produced and 100% free of animal derived raw materials and organic solvent residues. Animal derived ingredients are not used for production and the protein levels are very low and endotoxin free. Production follows the criteria established in the European Pharmacopoeia (European Pharmacopeia). HA hydrogels were prepared using Glycosil (C.) (II: (C.))

HA, ESI BIO-CG313) thiol-modified HA, which can be used to trigger Glycosil formation of disulfides using polyethylene glycol diacrylate (PEGDA). Degassed water was used in 1% Phosphate Buffered Saline (PBS) or in our case in the stock column medium

Reverting to a 1% solution of thiolated HA. On reconstitution, it remains in the liquid state for several hours, but if exposed to oxygen, it may undergo some gelation. If Glycosil is treated with a crosslinking agent (such as PEGDA) that causes gelation to occur within minutes, there is no temperature or pH changeMore precise gelation occurs in the case of chemical gelation.

The level of crosslinking determines the consistency level and can be precisely defined by the ratio of thiol-modified HA to PEGDA. In previous studies, stem cell populations were tested in HA hydrogels of different consistency levels, and it was found that they remained antigenic and functional (i.e. in terms of migratory capacity) stem cells only at consistency levels of less than 200Pa²³. We used this finding to design an implant of hyaluronic acid hydrogel with a very soft layer and a thicker layer on the serosal side to form a barrier to migration in directions other than the target tissue, as well as to minimize adhesion from cells near the tissue. The 3 hydrogel patterns with different consistency levels are characterized in fig. 2, i.e. including a characterization of the direct measurement of the rheological properties. The thickest barrier of 10 × HA hydrogel (consistency 760Pa) is prepared in advance on a substrate and can be stored at ultra low temperature if necessary. At the time of surgery, donor cells were prepared in soft 1 × HA hydrogel (consistency ═ 60 Pa); placed in a thicker 10 × hydrogel (already on the substrate); and the patch is tethered to the target site. After tethering, use the disc with BD Micro-Fine^TMNORM-JECT 4010.200V0 plastic syringe with permanently attached needle IV coats or coats the serosal side of the implant with 2 XHA hydrogel (consistency 106 Pa).

Stress-controlled cone-plate rheometers (TA Instruments, AR-G2, 40mm cone diameter, 1 ° angle) were used to determine the large-scale rheological properties of the hydrogels. The gel was living polymerized on a rheometer while oscillating at a frequency of 1rad/s and a stress amplitude of 0.6Pa and the modulus was continuously monitored to inquire whether the crosslinking reaction was sufficiently complete. Once equilibrated, the hydrogel can be subjected to an oscillation frequency sweep (stress amplitude: 0.6Pa, frequency range: 0.01-100 Hz). The viscoelastic (rheological) properties of the 3 hyaluronic acid hydrogels used are summarized in fig. 2.

The most commonly used donor cells were derived from transgenic H2B-GFP pigs as described above. They offer significant advantages for cell transplantation studies because all cells are labeled with GFP. The use of fluorescent proteins as molecular tags allows for tracking of donor cells in migration and implantation after transplantation. The fusion protein targets nucleosomes, thereby generating a nuclear/chromatin GFP signal. In the described implants, stem cells express GFP throughout the nucleus, but those lineages restricted to adult cell types may have GFP in the cytoplasm or nucleus. Note that cytoplasmic GFP levels were particularly high in the first week and decreased over time. This is because the implantation/invasion/integration process results in an effect on the cells, which may result in the presence of H2B-linked GFP in the cytoplasm. This does not mean that the cells are dying, but rather that they respond to high levels of MMPs and associated signaling as part of the remodeling region. Indeed, the GFP + cells examined were apparently viable and proliferating, all of which expressed various adult functions (e.g. albumin, HNF4a, AFP, insulin, glucagon or amylase).

As described in more detail in the characterization of implants, autofluorescence of both the basement (spring green) and lipofuscin (dark forest green) in mature hepatocytes poses a challenge in view of the wavelength overlap with GFP. Therefore, applicants used antibodies to GFP and secondary antibodies to antibodies with red fluorescent probes to convert the GFP + signal to pink or rose. This resulted in the stem cells being recognized as minicells with pink nuclei (a combination of blue DAPI staining of the nuclei and antibody-labeled rose GFP + labeling). Any donor cells that matured into hepatocytes were identified as having a combined color of light purple from green autofluorescence (lipofuscin), blue (DAPI), and rose (GFP) (fig. 4).

Porcine extrahepatic biliary tissue (gallbladder, common duct, hepatic duct) was obtained from transgenic pigs. Tissues were beaten with a sterile stainless steel hammer to remove parenchymal cells, taking care to maintain the intra-hepatic and extra-hepatic bile duct connections. The bile lines were then washed with "cell wash" buffer supplemented with antibiotics, 0.1% serum albumin and 1nM selenium (10)^-9M) sterile, serum-free basal medium. The biliary lines were then mechanically separated with a cross scalpel and aggregates were dispersed enzymatically in a cell suspension in RPMI-1640 supplemented with 0.1% Bovine Serum Albumin (BSA), 1nM selenium, 300U/ml type IV collagenase, 0.3mg/ml deoxyribonuclease (dnase) and antibiotics. Xiaoxiao (medicine for eliminating cough and asthma)The reaction is carried out at 32 ℃ for 30-60 minutes under continuous stirring. Most tissues required two rounds of digestion followed by centrifugation at 1100rpm at 4 ℃. The cell pellet was combined and resuspended in cell detergent. The cell suspension was centrifuged at 30G for 5 minutes at 4 ℃ to remove erythrocytes. The cell pellet was resuspended in cell detergent, filtered through a 40 μm nylon cell strainer (Becton Dickenson Falcon #352340), and washed with fresh cell detergent. Cell numbers were determined and viability was assessed using trypan blue. Cell viability greater than 90% -95% was routinely observed.

Mesenchymal cells collocated with BTSCs are subpopulations free of MHC antigens, with low side scatter, and can be identified as angioblasts (CD117+, CD133+, VEGF receptor + and CD31 negative), precursors of endothelium (CD133+, VEGF receptor + and CD31+) and precursors of astrocytes (CD146+, ICAM1+, VCAM +, α -smooth muscle actin (ASMA) + and vitamin a negative), these 3 subpopulations are collectively referred to as early lineage-stage mesenchymal cells (ELSMC), adult hepatocytes are associated with mature sinusoidal endothelium (CD31+ + +, IV protein +, VEGF receptor + and CD117 collagen negative) and biliary epithelial cells associated with mature astrocytes (ICAM-1+, ASMA +, vitamin a + +, I protein +).

The cell suspension was added to serum-free bank tower medium in a multi-well flat bottom cell culture plate (Corning #353043) and incubated at 37 ℃ for-one hour to promote adherence of mature mesenchymal cells. Mature mesenchymal cells adhere to the culture dish within 10-15 minutes, even if the medium is serum-free. The cells remaining in suspension were transferred to another dish and incubated again for up to one hour. This step is repeated to eliminate most of the mature mesenchymal cells. After removal of mature mesenchymal cells, the remaining floating cells were depleted by-2X 10⁵Individual cells/well were seeded in serum-free, bottom-of-the-pool column medium in Corning's ultra-low-adherence culture dish (Corning #3471) and incubated at 37 ℃ in a CO2 incubatorIncubate overnight. Organoids consisted of biliary stem cells (BTSCs) and overnight formed ELMSCs (fig. 1). These organoid cultures survived for weeks in the stock tower medium, especially in the case of medium supplementation (0.1%) with the soluble form of HA (Sigma); they may also be stored at ultra low temperatures as described below. We obtained-1.5X 10 from per gram of neonatal porcine biliary tissue⁷And (4) cells. In 6-hole ultra-low wall-mounted panels, we use 3-6X 10⁵Cells/well and incubated in serum-free depot column medium. Cells produce on average 6000 to 20,000 small organoids (-50-100 cells/organoid/well). For implants, we use at least 100,000 organoids ((ii))>10⁷Individual cells). Depending on the size of the substrate, applicants were able to increase the number of organoids in the implant up to 10⁸An organoid (i.e., -10)⁹Individual cells) or further embedded in-1 ml of soft hyaluronic acid hydrogel on a 3cm x 4.5cm substrate.

Isolated stem cell organoids were stored in CS10 at ultra low temperature, CS10 was an isotonic ultra low temperature storage buffer containing anti-freeze factor, dextran and DMSO (Bioliffe, Seattle, Washington; https:// www.stemcell.com/products/cryostor-CS10. html). Cell viability was further improved by 0.1% HA supplement (Sigma # 52747). Cryopreservation was performed using a cryoMedTM programmable freezer. The viability upon thawing is greater than 90%, and the cells after thawing are able to adhere to the wall, thereby expanding ex vivo and in vivo and producing the desired mature cells in vitro and in vivo.

Cell isolation and implant assembly are characterized in the schematic of fig. 1, the details of which are summarized in fig. 2. The implants were formed by using a substrate (table 1) on which the stem cell organoids embedded in a soft hyaluronic acid hydrogel were placed. These implants can be easily prepared in advance and maintained overnight in a petri dish in an incubator. The implant appeared stable at the target site throughout the experiment. Cryopreservation of organoids can be easily achieved, but is not necessarily the case when in soft hydrogels. This means that the organoids must be embedded in a soft hydrogel prior to surgery.

Surgery

Anesthesia was induced by administration of either a combination of intravenously injected ketamine/xylazine (2-3 mg/kg body weight each) or intramuscularly injected 20mg/kg ketamine plus 2gm/kg xylazine and maintained by administration of oxygen and isoflurane via a closed loop gas anesthesia unit.

The animals were placed in a dorsal position and the abdomen was excised from the xiphoid process to the pubic bone. The skin was prepared aseptically by alternating iodine swabbing and alcohol solutions. After entering the operating room, the preparation of the skin is repeated using sterile techniques, covering the area with a topical iodine solution, and then a sterile surgical drape. The surgeon uses appropriate aseptic techniques. A medial abdominal incision was made through the skin, through the subcutaneous tissue and the linea alba, starting from the xiphoid process and extending 8-12cm caudally. The left liver was split exposed and a 3 x 4.5cm patch implant containing 1 xha (-60 Pa) was applied to the abdominal surface of the liver, the embedded organoids were placed on a substrate containing 10 xha (-760 Pa) and the patch was placed in direct contact with the surface of the liver capsule. The patch implant was sutured to the liver using 4-6 simple, interrupted 4-0 polypropylene sutures. The exposed surface of the implant was then treated with 2ml of a 2 × HA hydrogel (-106 Pa) at a consistency level sufficient to allow it to coat or coat the serosal side of the implant; it is used to minimize adhesion from adjacent tissue. After placement of the surgical implant, the white line was closed by a simple continuous suture using 0-PDS. The white line was blocked with 2mg/kg 0.5% bupivacaine injected intramuscularly. The subcutaneous tissue and skin were closed with continuous 2-0PDS and 3-0Monocryl sutures, respectively. The tissue adhesive is placed on the skin surface.

The implant graft from the transgenic pig to the recipient is allogeneic, so immunosuppression is required. The immunosuppressive regimen used was one established by others. Starting 24 hours prior to surgery, all pigs received oral doses of the immunosuppressive drugs Tacrolimus (Tacrolimus) (0.5mg/kg) and mycophenolic acid (Mycophenolate) (500mg) twice daily. The drug was administered continuously throughout the experiment. These drugs can be easily administered to animals if they are mixed with foods that the animals like.

All animals were euthanized humanely by ketamine/xylazine sedation and isoflurane anesthesia at the indicated time points, followed by intravenous injection of a lethal dose of sodium pentobarbital. After confirmation of death, the cadavers were carefully dissected, removed and placed in cooled depot tower medium for transport to the laboratory. In addition to the liver, lungs, heart, kidneys and spleen were collected and fixed in 10% neutral formalin.

Characterization of implants

After 48+ hours of fixation, the tissue samples were placed in 70% ethanol in marker cassettes and processed in a LeicaASP300S tissue processor at 60 degrees for approximately 10 hours with long cycles. After the overnight treatment was completed, the samples were embedded using a LeicaEG1160 embedding machine. The mold is filled with wax and the sample is placed in the correct orientation for collection of the desired slices. The cartridge is cooled until the block and tissue sample can be removed from the mold as a unit. The blocks were cut into 5 micron sections using a Leica RM2235 microtome; the sections were floated in a water bath and placed on glass slides. Slides were allowed to air dry overnight before staining. Sections were stained with hematoxylin and eosin (H & E; reagents #7211 and #7111) or Masson's trichrome (Masson's trichrome stain: blue collagen kit #87019) using Richard Allan scientific histology products following the manufacturer's recommended protocol; this scheme has been programmed into a Leica Autostainer XL.

Tissues were embedded and frozen in OCT and snap frozen at-20 ℃ for frozen sectioning. The frozen sections were IHC stained according to the protocol described above. For immunofluorescence, frozen sections were thawed at room temperature for 1 hour and then fixed in 10% buffered formaldehyde, acetone or methanol according to antibody instructions. After fixation, sections were washed 3 times in 1% Phosphate Buffered Saline (PBS) and then blocked with 2.5% horse serum in PBS for 1 hour at room temperature. Primary antibody diluted in 10% goat serum in PBS was added and incubated overnight at 4 ℃. The following morning, sections were washed 3 times with PBS and incubated with secondary antibody diluted in 2.5% horse serum in PBS for 2 hours at room temperature. Images were acquired using a Zeiss CLSM 710 spectral confocal laser scanning microscope (Carl Zeiss microcopy). The antibodies are listed in table 3.

For the images in FIG. 5, sections (3 μm) were stained with hematoxylin-eosin and sirius red according to standard protocols for immunohistochemistry endogenous peroxidase activity was blocked by incubation for 30min in methanol hydrogen peroxide (2.5%), as described by the supplier, the sections were incubated at room temperature with primary antibodies (pan keratin, Dako, code: Z0622, dilution: 1: 100; Sox9, Millipore, code: AB5535, dilution: 1:200) overnight at 4 ℃, the samples were washed twice with PBS for 5min, with biotinylated secondary antibodies (LSA B + System-HRP, code K0690; Dako, Glostrip, Denmark, then with streptavidin-HRP (B + System-HRP), code K0690, and the sections were examined by staining with a confocal microscope (Involu) with a fluorescent microscope (Involu-ELISA, Involu-20) and examined by staining with a fluorescent staining procedure with a rabbit-rabbit staining procedure (Involuol) as well as a staining procedure, staining with a staining procedure No. 20, staining with a staining procedure No. 5-20, staining procedure No. 5, No. 4, and No. 5, No. 4, No. 5, No. 4, No. 5, No. 1, No. 4, No. 1, No. 4, No. 1, No. 5, No. 1, No. 4, No. 1, No. 4, No. 1, No. 4, No.

Frozen sections are problematic in view of the high autofluorescence of hepatocytes (lipofuscin) and the fluorescence of the Seri-Silk substrate. Applicants have achieved greater success by preparing paraffin sections and staining GFP with rabbit polyclonal antibodies to GFP (Novus Biologicals, NE 600-308); the rabbit anti-GFP antibody was used in combination with a donkey anti-rabbit IgG H & L secondary antibody (Alexa Fluor 568; ab175470, Invitrogen), while the donkey anti-goat IgG Alexa Fluor 488 antibody was used to exclude non-specific staining of liver autofluorescence. Autofluorescence was reduced by quenching with a dye including trypan blue. Trypan blue was dissolved in PBS at 0.4% for tissue/cells. This significantly reduces the background.

Total RNA was extracted from organoids or implants using trizol (invitrogen). First strand cDNA synthesized using Primescript 1 st strand cDNA Synthesis kit (Takara) was used as a template for PCR amplification. Quantitative analysis of mRNA levels was performed using Faststart Universal Probe template (Roche Diagnostics) with the ABI PRISM 7900HT sequence detection System (Applied Biosystems). Primers were designed at the Universal Probe library assay Design Center (Universal Probe library Design Center) (Roche Applied Science). The primer sequences are listed in table 4. The primers were annealed at 50 ℃ for 2min and treated at 95 ℃ for 10min, followed by 40 cycles of 95 ℃ (15s) and 60 ℃ (1 min). Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is commonly used as a control and standard.

RNA was purified from cells using Qiagen RNeasy kit and RNA Integrity (RIN) analysis was performed using Agilent2000 bioanalyzer. cDNA libraries were generated using Illumina TruSeq standard mRNA preparation kit and sequenced on Illumina HiSeq 2500 platform. Two samples per lane were sequenced, and for all samples (one flow cell), these samples occupied a total of 8 lanes. Quality control analysis was done using FastQ. Mapping of sequence reads to the human genome (hg19) was performed by mapspice 2 using default parameters. Transcript quantification was performed by RSEM analysis, and DESeq was used to normalize gene expression and identify differentially expressed genes. Mapscice 2 was also used to detect candidate fusion transcripts. The fusion identification is based on the depth and complexity of the reads that span the candidate fusion connection points. Pearson's correlation analysis was used to compare gene expression profiles and hierarchical clustering was performed in R. Hierarchical clustering was performed according to the differential stabilization transformation provided in the DESeq package. The Pathway enrichment Analysis was performed using Ingenity Path Analysis (IPA) software. Differential gene expression analysis was performed only on genes with minimum mean normalized counts >50 in at least one category.

The Student's two-tailed t-test was used to calculate statistically significant differences between samples, with results expressed as mean ± Standard Deviation (SD). P values less than 0.05 were considered statistically significant.

Results

In previous studies on injection implantation, it was found that implantation required co-transplantation of epithelial cells with a suitable mesenchymal cell partner for their lineage stage for liver stem cells and biliary stem cells, these mesenchymal cells consisted of angioblasts (CD117+, CD133+, VEGFr +, CD31 negative) and their direct progeny, precursors of endothelium (CD133+, VEGFr +, CD31+, weweber's factor +) and precursors of stellate cells (CD146+, ICAM-1+, α -smooth muscle + (ASMA), vitamin a negative).

In previous studies, applicants achieved the isolation of matched epithelial and mesenchymal cell phases using multi-parameter flow cytometry to determine the ratios of lineage stage partners of epithelial and mesenchymal cells in cell suspensions, and then use those ratios by immunoselection of the cells. In these studies, applicants found that it was more effective to cull the cell suspension of mature mesenchymal cells by repeating the panning procedure and then culturing the remaining cell suspension on low-adherence culture dishes and in serum-free bank media for 6-8 hours. Organoids self-assemble, containing approximately 50-100 cells per aggregate. Marker analysis indicated that BTSCs collocated with ELSMC (fig. 1). As summarized in the schematic illustration of fig. 1A, they can be used immediately or cryopreserved under defined conditions previously determined and thawed as needed for implantation. Immunofluorescence (IF), qRT-PCR and RNA-seq were used to characterize the organoids of BTSC/ELSMCs and showed classical behavior of expressing BTSC (FIG. 1) and ELSMCs (data not shown). BTSCs in organoids do not express mature liver or pancreas genes, but express low levels of pluripotency genes (e.g., OCT4, SOX2) and endoderm stem cell genes (e.g., EpCAM, SOX 9, SOX17, PDX1, LGR5, CXCR4, MAFA, NGN3, and NIS). Representative qRT-PCR assays confirmed findings from IF and from IHC against cells prior to transplantation (fig. 1D). IHC assays indicate that more primitive cells (e.g., cells expressing pluripotency genes) are distributed in the interior of organoids and in the peripheral, late maturation lineage (e.g., EpCAM or albumin expressing cells) (fig. 1C).

The results from the patch implants were compared to those from the injection implants by a previously established method consisting of cell injection and localized to the site by triggering hyaluronic acid gelation within minutes using polyethylene glycol diacrylate (PEGDA). Injection of the implant into the porcine liver parenchyma resulted in essentially 100% implantation, but had minimal (if any) migration and integration into host tissues slowly occurred over several weeks (data not shown). These findings are similar to those previously observed with injected implants of liver stem cells¹⁷. Injection of the implant through the large tube into the mesentery near the hepatic duct/portal branch immediately caudal to the liver lobes was feasible, but resulted in smaller ones being blocked by swelling of the HA hydrogel and resulted in cholestasis (fig. 13). The success of patch implants has given us up further work with injection implantation strategies.

The composition of the implant against stem cells involved conditions using Hyaluronic Acid (HA) hydrogel with 3 distinct layers, where the precise concentration of HA and PEGDA was used to achieve consistency levels assessed by rheometry (fig. 2C). Donor cells were embedded in a soft HA layer (-100 Pa) and placed against the liver/pancreas surface; at this pointIn these studies, the soft hydrogel maintained the dry state²³Prove important for implantation. This layer is placed on top of a thick (10 ×; -700 Pa) HA layer, prepared in advance on a substrate and acting as a barrier to migration. The patch is attached to the target site using sutures or surgical glue. The 2 xha hydrogel is soft enough (consistency ═ 200Pa) to allow coating or application to the serosal surface of the implant at the time of surgery and to minimize adhesion near the tissue.

The patch implant is placed on the surface of the liver, i.e. the surface of the gleason's capsule or pancreatic capsule, and attached on the corners by sutures or by surgical glue (fig. 2F). The consistency of Seri-Silk results in the implant being placed at the site of least flexure and away from the site of greater mechanical force (e.g., near the septum). In an implant implanted in the pancreas, the implant is wedged between the duodenum and the pancreas.

An attempt was then made to forego the only patch implant variant after surgical removal of the capsule completely. Given the adverse effects of serum on donor cells, excessive bleeding avoids future use in hosts with altered hemostasis associated with liver failure, or even in normal hosts. Without such work to alter the organ capsule, the patch implant is easy to perform a surgical procedure.

A number of substrates were tried, with emphasis on the substrates used clinically in abdominal surgery (tables 1 and 2). In addition to Seri-Silk, can cause problems that can lead to their removal for further consideration. These problems include fragility (e.g., Seprafilm, Retroglyde); induction of necrosis or fibrosis and significant levels of adhesions (e.g., Surgisis, Vetrix); and Seri-Silk sponge type of severe adhesion formation or any substrate with an abdominal supplement of carboxymethyl cellulose ("belly jelly"). Of those tested, the SERI procedure Silk^24-26(Allergan, inc. irvine, CA) provides the best combination of mechanical support and minimal adhesion, the effect being further enhanced by application of 2 x HA to the serosal surface of SeriSilk after attachment to the target site. This product was a purified fibroin of silk moth silk and was developed by David Kaplan (Tuft's University, Boston, MA). Shen-Shi-an exercise for strengthening the muscles and jointsApplicants found it to be thick, a property that was found to be useful for surgical manipulation and placement on flat/thick organs such as the liver. The consistency makes it difficult to apply to sites that have significant bends or require flexibility. In addition, its consistency proved to be neutral with respect to maturation against donor cells, which finding made the substrate useful for patch implants. In the 3-week implants, the Seri-Silk was encapsulated by collagen bands, indicating a mild foreign body response. Evaluation of other candidate substrates (such as synthetic fabrics) is ongoing.

Evidence of remodeling in the first week after surgery was verified using trichrome staining (fig. 3, 7) or safranin O, which has a dye that stains collagen and other extracellular matrix components. Images of implants stained with trichrome (fig. 3A-B) were compared to images of the same sites and stained with hematoxylin/eosin (fig. 3C-D). The reconstruction of the Gleason's capsule and lobule occurred at 3 weeks in parallel with the absorption of HA. The band including the remodeled region was very large (fig. 3-5, 7).

Donor cells derived from transgenic GFP + pigs can be readily identified by GFP expression as determined by IHC assay. In the pancreas, donor cells were identified by green fluorescence. However, in the liver, the autofluorescence of lipofuscin in hepatocytes peaks at a wavelength that overlaps with the autofluorescence of GFP. Therefore, we identified donor cells in the liver with antibodies to GFP (rabbit anti-GFP antibody; Novus, NB600-308) and coupled to a secondary antibody with a red fluorescent probe (donkey anti-rabbit 555, Invitrogen) so that the donor cells had pink nuclei (red fluorescent probe plus blue DAPI). Host cells could be identified due to their blue nuclei (DAPI staining), but these host cells did not express GFP (fig. 4).

Liver lobules of mature hepatocytes were forest green of autofluorescence (lipofuscin) (fig. 4B). Donor GFP + cells that had matured into aggregates of hepatocytes were light purple with pink nuclei (fig. 4C), because of the combined color of red fluorescent probe from GFP, blue from DAPI, and autofluorescence dark green from lipofuscin, host or donor-derived hepatocytes clustered around host mesenchymal cells (endothelium, stellate cells) with bright yellow/green autofluorescence, we believe due to vitamin a in mature stellate cells (fig. 4C); IHC data for endothelial and stellate cells are not shown.

Within one week, patch implants of BTSC/ELSMC organoids resulted in remodeling of the organ capsule and adjacent leaflets, followed by pooling of host and donor cells (fig. 3-5, 7). The finger-like extension of the donor cells extends to the liver lobules of the host tissue; in parallel, the host cells extended to the HA of the implant (fig. 4). In the case of the pancreas, the implant was wedged between the pancreas and the duodenum, and one week after surgery, implantation of donor cells occurred in the submucosal bunloner glands of the pancreas and duodenum (fig. 6). Cell integration in the large liver (or pancreas) area is complete at 2 weeks, when the HA layer is mostly taken up; donor cells have lineages restricted to adult hepatic parenchymal fates (cholangioepithelial and hepatocyte fates) (fig. 5) or pancreatic fates (fig. 6).

At 3 weeks, the HA layer is completely absorbed, leaving only the basement, which correlates with the histological structure of the organ capsule and tissue near the capsule (FIGS. 3, 5, 6) or the reproduction of the pancreatic capsule and pancreatic histological structure (FIG. 6). in the pancreas, mature cells are identified by functional markers including insulin against pancreatic islet cells (β cells) and amylase against acinar cells.

At one week, the implantation efficiency was close to 100% for both the liver and the pancreas, as all identified donor cells were found to be viable and within the liver or pancreas; not in the remnants of the implant on the organ capsule; and with negligible or no evidence of ectopic cell distribution in other organs (e.g., lungs).

The migration rate of donor cells through the liver and through the pancreas in BTSC/ELSMC implants was demonstrated to be very high, resulting in donor cells in most regions of the organ (liver or pancreas) at the end of one week and uniformly dispersed cells throughout the tissue (liver/pancreas) at 2-3 weeks (fig. 3-6).

Elevated expression of various MMPs, enzymes known to solubilize extracellular matrix components and to be associated with cell migration, is associated with lysis and remodeling of the glehnson capsule (or pancreatic capsule) and adjacent liver lobules (or pancreatic tissue) and with mass implantation. Data from RNA-seq studies and IHC assays for MMP expressed by stem/progenitor cells and adult cells are summarized in fig. 7. BTSCs express high levels of multiple MMPs, including secreted forms (e.g., MMP2, MMP7) and membrane-associated forms (e.g., MMP14 and MMP 15). Precursors of ELSMC, endothelium and stellate cells also contribute to a variety of MMPs.

The findings from the RNA-seq data were confirmed by IHC assay for protein encoded by MMP gene (fig. 7). The IHC assay confirms the presence of secreted forms of MMPs (e.g., MMP1, MMP2, MMP7, MMP9), especially in the remodeling region. Protein expression of MMP1 is present in BTSC/ELSMC organoids and in the remodeled region of implants; however, the existing database of RNA-seq discovery results does not include MMP1 due to the lack of annotated species of porcine MMP1 to be used for analysis. Therefore, its expression was recognized based on IHC assay.

The variables that lead to donor cell differentiation led to silencing of MMP expression, particularly in the secreted form, and in parallel the loss of potential for engraftment and migration (data not shown). These factors include serum, various soluble regulatory signals (growth factors, cytokines, hormones) known to affect the differentiation of donor cells, extracellular matrix components (whether in the hydrogel or in the basement (especially collagen type I-containing basement)), and the consistency of the HA hydrogel (i.e., Pa levels). Fibrosis of the implant occurs if the differentiation of the ELSMC preferentially progresses to the matrix; the implant retained viable cells and tissue if advanced to the endothelium, but remained on the surface of the organ capsule (data not shown).

Organoids of BTSC/ELSMC have proven to be the most successful arrangement for cells for implantation. In the past, we used unique surface antigens to co-transplant epithelial-mesenchymal partners by immunoselection of the epithelial-mesenchymal partner from a cell suspension by flow cytometry, and then mixed the epithelial-mesenchymal partners according to the ratio present in the cell suspension of freshly isolated tissue¹⁷. Here, we have found that, in this case,self-selection of mature mesenchymal cells as organoids after their removal by panning proves to be more efficient and effective in establishing lineage-appropriate epithelial-mesenchymal partners (with associated paracrine signaling for the implant) and in generating organoids under defined (serum-free) conditions (enabling their easy and safe cryopreservation).

The initial design of the implant involves mixing the cells with a suitable biomaterial that can be rendered insoluble and keep the cells localized to the target site. For implants, the ideal biomaterial HAs been shown to be a non-sulfated or minimally sulfated glycosaminoglycan (GAG), such as Hyaluronic Acid (HA), present in all stem cell niches, the receptor for HA having the classic stem cell trait. Maintaining the cells as stem/progenitor cells optimizes the expression of secreted and membrane-associated MMPs that are effective for implantation.

Evidence of the implantation process is particularly evident in the remodeled region, which occurs at the interface of the implant and host tissue. To verify the findings of remodeling, trichrome staining with dyes staining extracellular matrix components and safranin O were used and analyzed in parallel with hematoxylin/eosin stained adjacent sections (fig. 3, 7). It demonstrates remodeling of the organ capsule and adjacent tissue within one week after surgery. At 3 weeks post-surgery, these assays indicated histological reconstruction of organ capsules and normal tissue after HA clearance. The remodelling zone was very large (figures 3, 7), especially one week post surgery, and was shown to be involved in multiple forms of MMP (figure 7).

Although there are many sources and types of HA, the most useful is the thiol-modified HA established by Glenn Prestwich (University of utah, Salt Lake City, UT), which can be triggered by PEGDA to form hydrogels with precise biochemical and mechanical properties. These properties of HA confer perfect elasticity, allowing an implant to capture all soluble signals in blood, lymph or interstitial fluid, and minimizing maturation of donor cells until implantation and migration occur. The ability to alter rheological factors by simple changes in HA and PEGDA concentrations HAs been suggested in directing cell migration and minimizing adhesionProviding additional advantages. Soft HA hydrogels (i.e., hydrogels that mimic properties in the stem cell niche) are allowed to express stem/progenitor cell-associated MMP libraries. Therefore, the mechanical properties of HA (which have been studied for many years in terms of the function of skeletal tissue) are also important in managing the implantation strategy²³。

Patch implants containing stem/progenitor cells resulted in the surprising phenomenon of "thawing" of the implant into the tissue within a few days, followed by a combination of donor and host cells, and a distribution of cells over a large area of the organ over one to two weeks. Thereafter, donor cell maturation and organ capsule restoration occurred in parallel with tissue clearance of HA.

The implantation and integration processes are associated with the expression of a variety of MMPs, a family of calcium-dependent, zinc-containing endopeptidases that degrade extracellular matrix components. Using RNA-seq studies, we found patterns of stem/progenitor cell-associated MMPs consisting of high levels of secreted forms (e.g., MMP2, MMP7) and membrane-associated forms (e.g., MMP14, MMP 15). IHC assays indicate that protein levels of secreted MMPs (e.g. MMP1, MMP2, MMP7) were found to be abundantly expressed in the remodeled region (figure 7). Conditions leading to donor cell differentiation (soluble growth factors, cytokines, serum, matrix components, mechanical forces) result in a reduction of MMPs (especially secreted forms) and elimination of the parallel implantation process.

The biomaterial of the implant (HA in particular) HAs been shown to maintain the dry character of the cells ex vivo and in vivo. Since the implant does not contain known signals that can trigger fate determination, donor cell maturation is a finding of a unique adult fate, depending on whether the implant is placed on the liver or pancreas, suggesting that the local microenvironment of the host tissue is a logical source of relevant factors for the maturation process.

A large number of implantable cells (>10⁸) And depends on the size of the implant, the number of cells and the secreted MMP pool associated with the plasma membrane. These findings are associated with a limited number of cells (e.g., 10) that can be used for vascular delivery or injection implantation⁵-10⁶) The opposite is true.

Patch implants are a safety strategy by which large numbers of cells can be transplanted into solid organs, including internal organs, and may prove useful for the treatment of patients, especially where implantation can occur adequately in disease conditions. Although there are the following problems: in the case of fibrosis of the tissue or the effects of cirrhosis, abnormal implantation may occur. Accordingly, provided herein are embodiments of determining the efficacy of a patch implant for a method aspect.

Example 2: treatment of liver disease.

This example describes one exemplary method of treating a subject having a liver disease or disorder using a patch implant. Donor cells were prepared as organoids of biliary stem cells (BTSCs), liver and pancreas precursors, aggregates of Early Lineage Stage Mesenchymal Cells (ELSMCs) consisting of angioblasts and their early lineage stage progeny, precursors of endothelium and precursors of stellate cells as described herein. The BTSC/ELSMC organoids were embedded in a soft hyaluronic acid hydrogel (<200Pa) placed on a substrate tethered to a target site of the liver of a subject.

Following administration of the patch implant, the subject is monitored for improvement in liver function. Commonly used tests to examine liver function include, but are not limited to, alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), albumin, and bilirubin tests. The ALT and AST tests measure enzymes released by the liver in response to injury or disease. The albumin and bilirubin test measures how the liver produces albumin (protein) and how bilirubin is processed (waste product of the blood). It is expected that after about 2 weeks to about 36 weeks, an improvement in liver function will be detected. The improvement is determined by detecting an improvement in the value of one or more liver function tests relative to the value prior to implant administration and/or an improvement or amelioration of one or more symptoms of the liver disease or disorder.

Example 3: treatment of pancreatic diseases.

This example describes one exemplary method of treating a subject having a pancreatic disease or disorder using a patch implant. Donor cells were prepared as organoids of biliary stem cells (BTSCs), aggregates of Early Lineage Stage Mesenchymal Cells (ELSMCs) consisting of hemangioblasts and their early lineage stage progeny, precursors of endothelium and precursors of stellate cells as described herein. The BTSC/ELSMC organoids were embedded in a soft hyaluronic acid hydrogel (<200Pa) placed on a substrate tethered to a target site of the pancreas of the subject.

Following administration of the patch implant, the subject is monitored for an improvement in pancreatic function. Commonly used tests to examine pancreatic function include, but are not limited to, blood tests for levels of pancreatic enzymes amylase and lipase, direct pancreatic function tests after administration of secretin or cholecystokinin, fecal elastase tests, CT scans with contrast dyes, abdominal ultrasound, Endoscopic Retrograde Cholangiopancreatography (ERCP), endoscopic ultrasound, and magnetic resonance cholangiopancreatography. It is expected that after about 2 to about 36 weeks, an improvement in pancreatic function will be detected. The improvement is determined by detecting an improvement in one or more pancreatic function tests relative to the value prior to implant administration and/or an improvement or amelioration of one or more symptoms of the pancreatic disease or disorder.

Example 4: treatment of kidney disease.

This example describes one exemplary method of treating a subject having a renal disease or disorder using a patch implant. Donor cells were prepared as organoids of biliary stem cells (BTSCs), aggregates of Early Lineage Stage Mesenchymal Cells (ELSMCs) consisting of hemangioblasts and their early lineage stage progeny, precursors of endothelium and precursors of stellate cells as described herein. The BTSC/ELSMC organoids were embedded in a soft hyaluronic acid hydrogel (<200Pa) placed on a substrate tethered to a target site of the subject's kidney.

Following administration of the patch implant, the subject is monitored for an improvement in renal function. Commonly used tests to examine pancreatic function include, but are not limited to, clinically relevant endpoints of renal function known in the art. It is expected that an improvement in renal function will be detected after about 2 weeks to about 36 weeks. The improvement is determined by detecting an improvement in the value of one or more renal function tests relative to the value prior to implant administration and/or an improvement or amelioration of one or more symptoms of the pancreatic disease or disorder.

Example 5: treatment of gastrointestinal disorders.

This example describes one exemplary method of using a patch implant to treat a subject having a disease or disorder of the gastrointestinal tract. Donor cells were prepared as organoids of biliary stem cells (BTSCs), aggregates of Early Lineage Stage Mesenchymal Cells (ELSMCs) consisting of hemangioblasts and their early lineage stage progeny, precursors of endothelium and precursors of stellate cells as described herein. The BTSC/ELSMC organoids are embedded in a soft hyaluronic acid hydrogel (<200Pa) placed on a substrate tethered to a target site of the intestine of a subject.

Following administration of the patch implant, the subject is monitored for an improvement in intestinal function. Commonly used tests to examine bowel function include, but are not limited to, clinically relevant endpoints of bowel function known in the art. It is expected that after about 2 weeks to about 36 weeks, an improvement in bowel function will be detected. The improvement is determined by detecting an improvement in the value of one or more bowel function tests relative to the value prior to administration of the implant and/or an improvement or amelioration of one or more symptoms of the gastrointestinal disease or disorder.

Specification accessory

Tables 1-4 Patch Grafts (Patch implants) 2018

Claims (47)

1. A patch implant for retaining and maintaining a mixed cell population, the patch implant comprising:

(a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population within or away from the patch implant; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in the direction of the substrate or barrier,

the patch implant is configured to maintain and maintain the mixed population while inhibiting differentiation or further maturation of the at least one early lineage stage cell type to a late lineage stage that is no longer capable of expressing membrane-associated and/or secreted MMPs.

2. The patch implant of claim 1, wherein the substrate comprises a porous mesh infused with a hydrogel.

3. The patch implant of claim 1, further comprising:

(c) a hydrogel superimposed on a serosal surface of the substrate, the serosal surface being opposite the surface contacting the mixed population.

4. The patch implant of claim 1, wherein the hydrogel of element (a) comprises one or more hyaluronic acids.

5. The patch implant of claim 2, wherein the hydrogel of element (b) comprises one or more hyaluronic acids.

6. The patch implant of claim 3, wherein the hydrogel of element (c) comprises one or more hyaluronic acids.

7. The patch implant of claim 1, wherein the culture medium comprises a depot tower medium or other medium that supports "dryness".

8. The patch implant of claim 1, wherein the mixed population comprises mesenchymal cells and epithelial cells.

9. The patch implant of claim 8, wherein the mesenchymal cells comprise early lineage mesenchymal cells (ELSMCs).

10. The patch implant of claim 9, wherein the ELSMC includes one or more of a hemangioblast, a precursor of endothelium, or a precursor of stellate cells or Mesenchymal Stem Cells (MSCs).

11. The patch implant of claim 8, wherein the epithelial cells comprise epithelial stem cells.

12. The patch implant of claim 8, wherein the epithelial cells comprise biliary stem cells (BTSCs).

13. A patch implant as in claim 8, wherein the epithelial cells comprise committed and/or mature epithelial cells.

14. A patch implant as claimed in claim 13, wherein the oriented and/or mature epithelial cells comprise mature parenchymal cells.

15. The patch implant of claim 14, wherein the mature parenchymal cells include one or more of hepatocytes, cholangiocytes, and islet cells.

16. The patch implant of claim 8, wherein both the mesenchymal cells and epithelial cells comprise stem cells.

17. The patch implant of claim 1, wherein the mixed population comprises autologous and/or allogeneic cells.

18. The patch implant of claim 1, wherein one or more cell types are genetically modified.

19. The patch implant of claim 1, wherein the substrate comprises a porous mesh, scaffold, or membrane.

20. The patch implant of claim 1, wherein the substrate comprises a non-porous material.

21. A patch implant according to claim 20, wherein the non-porous material is selected from silk, amnion, placenta, omentum, synthetic fabrics, derivatives of the foregoing, or combinations thereof.

22. The patch implant of claim 1, wherein the substrate has sufficient elasticity to withstand mechanical forces, is capable of tethering to a target organ or tissue, and has sufficient flexibility to tether to a position having a bend.

23. The patch implant of claim 1, wherein any biomaterial (other than hydrogel) may be employed, provided that the biomaterial is capable of retaining and maintaining the cell population and has rheological properties (e.g., viscoelasticity) sufficient to allow the cell population to migrate within or away from the patch implant.

24. A patch implant for retaining and maintaining a cell population, the patch implant comprising:

(a) a population of cells (optionally a population of cells of a single type) supported in a culture medium in a hydrogel or other biomaterial having rheological properties (e.g., viscoelasticity) sufficient to allow the cells to migrate within or away from the patch implant; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration (or provide a barrier to migration) of the population of cells in the direction of the substrate.

25. A covering or coating for a patch implant or tissue comprising a hydrogel or other biomaterial having sufficient viscoelastic and elastic properties to withstand mechanical forces, including such forces as from other tissues and organs.

26. A method of preventing adhesions involving or resulting from mechanical forces or contact from other organs and tissues, the method comprising covering or coating a surface with hydrogel or other comparable biological material.

27. A method of implanting cells into a target tissue, the method comprising contacting the target tissue with a patch implant comprising:

(a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population toward and into a target tissue; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population away from a target tissue and through the substrate or barrier.

28. The method of claim 27, further comprising allowing the cells contained in the patch implant to incorporate into the tissue.

29. The method of claim 27, wherein the target tissue is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, gastrointestinal tract, lung, prostate, breast, brain, bladder, spinal cord, skin, uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and skeletal tissue.

30. The method of claim 29, wherein the target tissue is liver tissue.

31. The method of claim 29, wherein the target tissue is pancreatic tissue.

32. The method of claim 29, wherein the target tissue is biliary tissue.

33. The method of claim 29, wherein the target tissue is a gastrointestinal tract tissue.

34. The method of claim 29, wherein the target tissue is kidney tissue.

35. The method of claim 29, wherein the target tissue is an organ.

36. The method of claim 35, wherein the organ is an organ of the musculoskeletal system, digestive system, respiratory system, urinary system, female reproductive system, male reproductive system, endocrine system, circulatory system, lymphatic system, nervous system, or integumentary system.

37. The method of claim 35, wherein the organ is selected from the group consisting of liver, pancreas, biliary system, thyroid, thymus, intestine, lung, prostate, breast, brain, bladder, spinal cord, skin, uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and bone.

38. A method of implanting cells into a target tissue, the method comprising contacting the target tissue with a patch implant comprising:

(a) a population of cells, including one having an early lineage stage, the population comprising a single type or multiple types of cells supported in a culture medium in a hydrogel or other biological material having rheological properties (e.g., viscoelasticity) sufficient to allow the cells of the population to migrate within or away from the patch implant; and

(b) a substrate comprising a biocompatible, biodegradable material having rheological properties (e.g., viscoelasticity) sufficient to inhibit migration (or provide a barrier to migration) of cells of the population in the direction of the substrate,

the patch implant is configured to hold and maintain the cell population while inhibiting differentiation or further maturation of the one population having an early lineage stage to a later lineage stage.

39. The method of claim 38, wherein the one population with an early lineage stage is capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs).

40. A method of treating a subject having a liver disease or disorder, the method comprising contacting the liver of the subject with a patch implant comprising: (a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population toward and into a target tissue; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in a direction away from the target tissue and through the substrate or barrier, an

Allowing the cells contained in the patch implant to bind into the liver, thereby restoring a certain liver function.

41. The method of claim 40, wherein the liver disease or disorder is liver fibrosis, cirrhosis, hemochromatosis, liver cancer, biliary atresia, nonalcoholic fatty liver disease, hepatitis, viral hepatitis, autoimmune hepatitis, fascioliasis, alcoholic liver disease, α 1-antitrypsin deficiency, glycogen storage disease type II, transthyretin-associated hereditary amyloidosis, Gilbert's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Padd-Guillai syndrome, liver trauma, or Wilson's disease.

42. A method of treating a subject having a pancreatic disease or disorder, the method comprising contacting a pancreas of the subject with a patch implant comprising: (a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population toward and into a target tissue; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in a direction away from the target tissue and through the substrate or barrier, an

Allowing the cells contained in the patch implant to bind into the pancreas, thereby restoring certain pancreatic function.

43. The method of claim 42, wherein the pancreatic disease or disorder is diabetes, pancreatic exocrine insufficiency, pancreatitis, pancreatic cancer, Oddi (Oddi) sphincter dysfunction, cystic fibrosis, pancreatic division, cricoid pancreas, pancreatic trauma, or pancreatic ductal hemorrhage.

44. A method of treating a subject having a disease or disorder of the gastrointestinal tract, the method comprising contacting one or more intestines of the subject with a patch implant comprising: (a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population toward and into a target tissue; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in a direction away from the target tissue and through the substrate or barrier, an

Allowing the cells contained in the patch implant to bind into the intestine, thereby restoring certain intestinal function.

45. The method of claim 44, wherein the gastrointestinal disease or disorder is gastroenteritis, gastrointestinal cancer, ileitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, peptic ulcer disease, celiac disease, fibrosis, vascular dysplasia, Hirschsprung's disease, pseudomembranous colitis, or gastrointestinal trauma.

46. A method of treating a subject having a kidney disease or disorder, the method comprising contacting one or more kidneys of the subject with a patch implant comprising: (a) a mixed population of two or more cell types, at least one type of which is in an early lineage stage capable of expressing membrane-associated and/or secreted Matrix Metalloproteinases (MMPs), supported in a culture medium in a hydrogel having sufficient viscoelastic properties to allow migration of the mixed population toward and into a target tissue; and

(b) a substrate comprising a biocompatible, biodegradable material having sufficient viscoelastic properties to inhibit migration of the mixed population in a direction away from the target tissue and through the substrate or barrier, an

Allowing said cells contained in said patch implant to bind into said kidney, thereby restoring a certain kidney function.

47. The method of claim 46, wherein the kidney disease or disorder is nephritis, nephropathy, nephritic syndrome, nephrotic syndrome, chronic nephropathy, acute kidney injury, kidney trauma, cystic nephropathy, polycystic nephropathy, glomerulonephritis, IgA nephropathy, lupus nephritis, kidney cancer, Alport syndrome, amyloidosis, Goodpastel's syndrome, or Wegener's granulomatosis.

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